Carbon monoxide dependent guanylyl cyclase modifiers and methods of use

ABSTRACT

Disclosed herein are methods and associated compositions and medicaments directed generally to the control of cellular and neural activity and for selectively and controllably inducing the in vivo genetic expression of one or more naturally occurring genetically encoded molecules in mammals. More particularly, the present invention selectively activates or derepresses genes encoding for specific naturally occurring molecules such as proteins or neurotrophic factors and induces the endogenous production of such naturally occurring compounds through the administration of carbon monoxide dependent guanylyl cyclase modulating purine derivatives. The methods of the present invention may be used to affect a variety of cellular and neurological functions and activities and to therapeutically or prophylactically treat a wide variety of neurodegenerative, neurological, cellular, and physiological disorders.

RELATED APPLICATIONS

[0001] The present invention is a continuation-in-part of co-pendingapplication Ser. No. 08/488,976, filed Jun. 8, 1995, which is acontinuation-in-part of co-pending application Ser. No. 08/280,719,filed Jul. 25, 1994.

FIELD OF THE INVENTION

[0002] The present invention relates in general to the control ofcellular and neural activity and to the treatment of cellular and neuraldisorders. More particularly, the present invention is directed tomethods and associated compositions and medicaments for the modificationof mammalian cellular and neural activity through the administration ofcarbon monoxide dependent guanylyl cyclase modulating purine derivativeswhich selectively and controllably induce the in vivo genetic expressionof naturally occurring genetically encoded molecules includingneurotrophic factors. The methods, compositions, and medicaments of thepresent invention may be used to affect a variety of cellular andneurological activities and to therapeutically or prophylactically treata wide variety of physiological, neurodegenerative, and neurologicaldisorders.

BACKGROUND OF THE INVENTION

[0003] The evolution of the central nervous system in mammals was anatural response to an increasingly complex environment requiringsolutions to difficult problems. The resulting structure is an intricatebiochemical matrix that is precisely controlled and attenuated throughan elaborate system of chemically modulated regulatory pathways. Throughan elaborate series of highly specific chemical reactions, thesepathways oversee and direct every structural and operational aspect ofthe central nervous system and, through it, the organism itself.Normally the complex interplay of the various control systems cooperatesto produce a highly efficient, versatile central nervous system managedby the brain. Unfortunately, when the biochemical matrix of the centralnervous system is damaged, either through age, disease or other reasons,the normal regulatory pathways may be incapable of effectivelycompensating for the loss. In such cases it would be highly desirable tomodify or supplement the neural mechanisms to prevent or compensate forsuch disorders. That is the focus of the present invention.

[0004] More specifically, the mammalian brain is composed ofapproximately ten billion nerve cells or “neurons” surrounded by a evengreater number of support cells known as neuroglia or astrocyte cells.Neurons, like other cells of the body, are composed of a nucleus, acytoplasm and a surrounding cell membrane. However, unlike other cells,neurons also possess unique, fiberlike extensions allowing eachindividual nerve cell to be networked with literally thousands of othernerve cells to establish a neural infrastructure or network.Communication within this intricate network provides the basis for allmental processes undertaken by an organism.

[0005] In each nerve cell, incoming signals are received by neuralextensions known as “dendrites” which may number several thousand pernerve cell. Similarly, neural information is projected along nerve cell“axons” which may branch into as many as 10,000 different nerve endings.Together, these nerve cell axons and dendrites are generally termed“neurites” through which each individual neuron can form a multitude ofconnections with other neurons. As a result, the number of possibleneural connections in a healthy brain is in the trillions, giving riseto tremendous mental capacity. Conversely, when the connections withinthe neural network break down as nerve cells die or degenerate due toage, disease, oxidative stress, or direct physical insult, the mentalcapacity of the organism can be severely compromised.

[0006] The connection of the individual axons with the dendrites or cellbodies of other neurons takes place at junctions or sites known as“synapses.” It is at the synapse that the individual neurons communicatewith each other through the flow of chemical messengers across thesynaptic junction. The majority of these chemical messengers, or“neurotransmitters,” are small peptides, catecholamines or amino acids.When the appropriate stimulus is received by a neural axon connection,the neurotransmitters diffuse across the synapse to the adjacent neuron,thereby conveying the stimulus to the next neuron along the neuralnetwork. Based upon the complexity of the information transferredbetween the nerve cells, it is currently believed that between 50 and100 distinct neurotransmitters are used to transmit signals in themammalian brain.

[0007] Quite recently, it was discovered that nitric oxide (NO) andcarbon monoxide (CO) may function as neurotransmitters. These gaseousmolecules appear to participate in a number of neuronal regulatorypathways affecting cell growth and interactions. In the brain, as wellas in other parts of the body, CO is produced by the enzyme “hemeoxygenase II” (HO). Whether produced from the HO enzyme or from othersources, it is believed that when CO diffuses into a neuron it induces arise in a secondary transmitter molecule known as “cyclic guanosinemonophosphate” (cGMP), by modulating an enzyme known as “guanylatecyclase” or “guanylyl” cyclase. Thus, CO acts as a signaling molecule inthe guanylyl cyclase regulatory pathway. The resultant increase in cGMPlevels appears to modify several neurotropic factors as well as otherneuronal factors which may induce, promote or modify a variety ofcellular functions including cell growth, protection, and intercellularcommunication.

[0008] Neurotrophic factors are molecules that exert a variety ofactions stimulating both the development and differentiation of neuronsand the maintenance of cellular integrity and are required for thesurvival and development of neurons throughout the organism's lifecycle. Generally, neurotrophic factors may be divided into two broadclasses: neurotrophins and pleiotrophins. Pleiotrophins differ from theneurotrophins in that they lack a molecular signal sequencecharacteristic of molecules that are secreted from cells and they alsoaffect many types of cells including neurons. Two effects ofneurotrophic factors are particularly important: (i) the prevention ofneuronal death and (ii) the stimulation of the outgrowth of neurites(either nascent axons or dendrites). In addition, it appears thatCO-induced neurotrophic factors may reduce the membrane potential ofnerve cells making it easier for the neurons to receive and transmitsignals.

[0009] Many of today's researchers believe that memory is associatedwith the modification of synaptic activity, wherein the synapticconnections between particular groups of brain neurons becomestrengthened or facilitated after repeated activation. As a result,these modified connections activate much easier. This type offacilitation is believed to occur throughout the brain but may beparticularly prominent in the hippocampus, a brain region which iscrucial for memory. The stimulation of neuronal pathways within thehippocampus can produce enhanced synaptic transmission through thesepathways for many days following the original stimulation. This processis known as long term potentiation (LTP).

[0010] More particularly, long term potentiation is a form ofactivity-dependent synaptic electrical activity that is exhibited bymany neuronal pathways. In this state, generally accepted as a type ofcellular memory, nerve cells are more responsive to stimulation.Accordingly, it is widely believed that LTP provides an excellent modelfor understanding the cellular and molecular basis of synapticplasticity of the type that underlies learning and memory invertebrates, including man.

[0011] NO and CO are currently the leading candidates for messengersubstances that facilitate LTP because inhibitors of these compoundsretard the induction of potentiation. The ability to modify neuralactivity and to increase the ease of LTP using these or other signaltransducers could potentially increase learning rates and cognitivepowers, possibly compensating for decreased mental acuity. Prior to thepresent invention, there were no known methods or agents which couldoperate on the cellular level in vivo to reliably modify cellular andneural regulatory pathways so as to facilitate the LTP of neurons.

[0012] In contrast to the enhanced mental capacity provided by long termpotentiation, mental functions may be impeded to varying degrees whenthe neuronal network is disrupted through the death or dysfunction ofconstituent nerve cells. While the decline in mental abilities isdirectly related to the disruption of the neural network, it isimportant to remember that the disruption is occurring on an individualcellular level. At this level the deleterious effects associated withneuronal disruption may be brought about by any one of a number offactors including neurodegenerative diseases and disorders, heartattack, stroke, aging, trauma, and exposure to harmful chemical orenvironmental agents.

[0013] Among the known neurological diseases which adversely impactneuronal function are Alzheimer's disease and related disorders,Parkinson's disease, motor neuropathic diseases such as AmyotrophicLateral Sclerosis, cerebral palsy, multiple sclerosis, and Huntington'sdisease. Similar problems may be brought about by loss of neuronalconnectivity due to normal aging or through damage to neurons fromstroke, heart attack, or other circulatory complications. Directphysical trauma or environmental factors including chemical agents,heavy metals and the like may also provoke neuronal or cellulardistress, dysfunction, or death.

[0014] Accumulated cellular damage due to oxidative free radicals isbelieved to be one of the critical factors in a variety of cellular andneurodegenerative diseases including Amyotrophic Lateral Sclerosis,Parkinson's disease, Alzheimer's disease, cancer, and aging. Most cellspossess a variety of protective mechanisms that guard against cytotoxicfree radicals. For example, high levels of glutathione may protectagainst free radical oxidation. Neurons are deficient in thisantioxidant source.

[0015] Whatever the cause of the neural disorder or dysfunction, thegeneral inability of damaged nerve cells to undergo substantial regrowthor regeneration under natural conditions has led to the proposal thatneurotrophic factors be administered to nerve cells in order to helprestore neuronal function by stimulating nerve growth and function.Similarly, stimulating neuritogenesis, or the growth of neurites, byadministering neurotrophic factors may contribute to the ability ofsurviving neurons to form collateral connections and thereby restoreneural function.

[0016] At present, prior art techniques and compounds have not beeneffective or practical to directly administer neurotrophic factors to apatient suffering from a neural disorder. In part, this is due to thecomplex molecular interaction of the neurotrophic factors themselves andto the synergistic regulation of neural cell growth and neuritogenesis.Neurotrophic factors are the result of a long chemical cascade which isexquisitely regulated on the molecular level by an intricate series oftransmitters and receptors. Accordingly, neuronal cells are influencedby a concert of different neurotrophic factors, each contributing todifferent aspects of neuronal development at different times.Neurotrophic factors are, effectively, the tail end of this cascade andthus are one of the most complex components of the regulatory pathway.As such, it was naive for prior art practitioners to assume that theunattenuated administration of single neurotrophic factors at randomtimes (from the cells viewpoint) could substantially improve cellactivity or regeneration. In contrast, modification of the regulatorypathway earlier in the cascade could allow the proper growth factors tobe produced in the correct relative amounts and introduced into thecomplex cellular environment at the appropriate time.

[0017] Other practical considerations also preclude the prior art use ofneurotrophic factors to stimulate the regeneration of the neuronalnetwork. Neurotrophic factors (including neurotrophins andpleiotrophins) are large proteins and, as such, are not amenable tonormal routes of medical administration. For example, these proteinscannot be delivered to a patient or subject orally as the patient'sdigestive system would digest them before they reached the target neuralsite. Moreover, due to their relatively large size, the proteins cannotcross the blood brain barrier and access the most important neurologicalsite in the body. Alternatively, the direct injection of neurotrophicfactors into the brain or cerebrospinal fluid crudely overcomes thisdifficulty but is fraught with technical problems of its own which havethus far proven intractable. For example, direct infusion of knownneurotrophins into the brain has proven impractical as it requiresadministration over a period of years to provide therapeuticconcentrations. Further, direct injection into the brain has beenassociated with dangerous swelling and inflammation of the nerve tissueafter a very short period of time. Thus, as theoretically desirable asthe direct administration of neurotrophic factors to a patient may be,at the present time, it is unfeasible.

[0018] Accordingly, it is a general object of the present invention toprovide methods and associated compositions and medicaments foreffectively modifying mammalian cells, neurons, cellular activity, orneural activity to achieve a variety of beneficial results. Theseresults include protection against oxidative stress and damage by freeradicals and more generalized physiological responses such as reductionsin mammalian blood pressure.

[0019] Thus, it is another object of the present invention to providemethods and associated compositions and medicaments for treatingmammalian neurological diseases and cellular disorders.

[0020] It is yet another object of the present invention to providemethods and associated compositions and medicaments for inducing longterm changes in the membrane potential of a mammalian neuron.

[0021] It is still another object of the present invention to providemethods and associated compositions and medicaments for inducing the invivo physiological production and administration of genetically encodedmolecules and neurotrophic factors within cells.

[0022] It is a further object of the present invention to providemethods and associated compositions and medicaments for enhancing theneurotogenic effects of neurotrophic factors in a physiologicalenvironment.

SUMMARY OF THE INVENTION

[0023] These and other objects are accomplished by the methods,compositions, and medicaments of the present invention which, in a broadaspect, provide for the selective inducement of the in vivo geneticexpression and resultant production of naturally occurring geneticallyencoded molecules including neurotrophic factors, and for themodification of cellular and neural activity through the treatment ofmammalian cells and neurons with at least one carbon monoxide dependent,guanylyl cyclase modulating, purine derivative.

[0024] As will be appreciated by those skilled in the art, the in vivoactivation or derepression of genetic expression and the exemplarymodifications of cellular and neural activity brought about by themethods, compositions, and medicaments of the present invention may beexpressed in a variety of forms or combinations thereof. For example,the treatment of a mammalian cell or neuron through the teachings of thepresent invention may result in the cell's direct self-administration ofthe in vivo expressed molecule(s) through the enhanced cellularproduction of various naturally occurring genetically encoded compounds,such as proteins and neurotrophic factors, or in the stimulation of theactivity of those compounds and their subsequent effect on naturallyoccurring cellular or neuronal metabolism, function, development, andsurvival. These subsequent effects can include protection from freeradical oxidation and cellular destruction, stabilization of cellreceptors against other factors, the endogenous production of carbonmonoxide and antioxidant compounds, and even reductions in bloodpressure via carbon monoxide activated cellular mechanisms. The methodsand medicaments of the present invention may also stimulate the growth,development and survival of the cell or neuron directly without thedeleterious effects of prior art factor methodology. Further, thepresent invention may be used to lower or change the membrane potentialof the cell, increasing its plasticity and inducing long termpotentiation.

[0025] Exemplary carbon monoxide dependent guanylyl cyclase modulatingpurine derivatives useful for practicing the present invention includeguanosine, inosine pranobex and4-[3-(1,6-dihydro-6-oxo-9-purin-9-yl)-1-oxopropyl]amino]benzoic acid(AIT-082). Unlike prior art compounds, these compounds may beadministered directly to a patient either orally or through injection orother conventional routes. These exemplary compounds are nontoxic andwill cross the blood-brain barrier as well.

[0026] In a further, more specific aspect, the methods and compositionsof the present invention may be used for the treatment or prophylacticprevention of neurological diseases and other cellular disorders,including those brought about by disease, oxidative stress, age, traumaor exposure to harmful chemical agents. By promoting the survival,growth and development of individual neurons and cells, the presentinvention facilitates the regeneration and development of the neuralnetwork and alleviates the manifestations of cellular and neuraldysfunction.

[0027] Of course, those skilled in the art will appreciate thatpharmaceutical compositions and medicaments may be formulatedincorporating effective concentrations of the carbon monoxide dependentguanylyl cyclase modifying purine derivatives of the present inventionalong with pharmaceutically acceptable excipients and carriers. Thesepharmaceutical compositions may be administered orally, transdermally,topically or by injection. Moreover, as the active agents used in themethods of the present invention can cross the blood-brain barrier, theydo not have to be injected or infused directly into the brain or centralnervous system.

[0028] In yet another aspect, the methods and compositions of thepresent invention may be used to induce long term changes in themembrane potential of a mammalian neuron. These long term potentiationchanges may lead to increased membrane plasticity with a correspondingenhancement of cellular memory. In turn, this enhanced cellular memorymay elevate the mental capacity of the subject leading to fasterlearning and increased retention of material.

[0029] Other objects, features and advantages of the present inventionwill be apparent to those skilled in the art from a consideration of thefollowing detailed description of preferred exemplary embodimentsthereof taken in conjunction with the data expressed in the associatedfigures which will first be described briefly.

BRIEF DESCRIPTION OF THE DRAWINGS

[0030]FIG. 1 is a graphical representation of murine plasmaconcentration following administration of the purine derivative AIT-082in accordance with the present invention;

[0031]FIG. 2 is a graphical representation of the effect of atropine, acholinergic antagonist, on memory enhancement in mice by the purinederivative AIT-082;

[0032]FIG. 3 is a graphical representation of nerve growth factormediated neurotogenic response in neuronal cells grown in vitro withvarious concentrations of the purine derivative AIT-082;

[0033]FIGS. 4A, 4B and 4C are graphical comparisons of the effects ofselective inhibitors and the purine derivative AIT-082 on nerve growthfactor mediated neurotogenic response; FIG. 4A shows the neurotogenicresponse of cells grown in the presence of methemoglobin, a carbonmonoxide scavenger; FIG. 4B shows the same response of cells grown inthe presence of methylene blue, a guanylyl cyclase inhibitor FIG. 4Cshows the response of cells grown in the presence of zinc protoporphyrinIX, a carbon monoxide scavenger;

[0034]FIGS. 5A and 5B are graphical comparisons of nerve growth factormediated neurotogenic response for cells grown in the presence of thepurine derivative AIT-082 and various concentrations of nitric oxideinhibitors;

[0035]FIG. 6 is a graphical comparison of cyclic GMP production inneuronal cells grown in culture with the purine derivative AIT-082 andwithout AIT-082;

[0036]FIG. 7 is a graphical representation of the effects of differentdoses of the purine derivative AIT-082 on learning as measured in SwissWebster mice using a winshift memory test;

[0037]FIG. 8 is a graphical comparison of the duration of action of thepurine derivative AIT-082 measured over time for single doses of 60mg/kg and 30 mg/kg;

[0038]FIG. 9 is a graphical comparison of learning abilities ofage-induced memory deficit Swiss Webster mice treated with the purinederivative AIT-082 and the drug physostigmine;

[0039]FIG. 10 is a graphical comparison of learning abilities ofage-induced memory deficit C57BL/6 mice treated with the purinederivative AIT-082 and the drug physostigmine;

[0040]FIG. 11 is a graphical comparison of age-induced memory deficitprophylaxis in mice treated with the purine derivative AIT-082 anduntreated mice;

[0041]FIGS. 12A and 12B are graphical comparisons of the production ofnerve growth factor by murine cortical astrocytes in response to theaddition of purine derivatives as measured using an ELISA assay; FIG.12A illustrates measured nerve growth factor concentrations for neuronsgrown in the presence of different concentrations of guanosinetriphosphate and FIG. 12B illustrates nerve growth factor concentrationsfor cells grown in the presence of various concentrations of guanosine;

[0042]FIGS. 13A and 13B are graphical comparisons of the production ofvarious neurotrophic factor mRNA by murine cortical astrocyte cellsgrown in the presence and absence of guanosine at different times; FIG.13A illustrates mRNA levels of nerve growth factor (NGF) and FIG. 13Billustrates mRNA levels of fibroblast growth factor (FGF);

[0043]FIGS. 14A, 14B and 14C are graphical comparisons of neurotogenicresponses to different concentrations of purine derivative in thepresence and absence of nerve growth factor; FIG. 14A illustratesneurotogenic response to various purine derivatives at differentconcentrations in the presence of nerve growth factor, FIG. 14Billustrates neurotogenic response in the absence of nerve growth factorand FIG. 14C illustrates neurotogenic response to individual purinederivatives and combinations of purine derivatives in the presence andabsence of nerve growth factor;

[0044]FIGS. 15A, 15B and 15C are graphical comparisons of nerve growthfactor mediated neurotogenic responses in neurons grown in the presenceof various concentrations of different purine derivatives; FIG. 15Aillustrates neurotogenic response to various concentrations of inosine;FIG. 15B illustrates the same neurotogenic response to variousconcentrations of hypoxanthine and FIG. 15C illustrates the neurotogenicresponse of neuronal cells exposed to different concentrations ofxanthine;

[0045]FIG. 16 is a graphical representation of nerve growth factormediated neuritogenesis measured for neuronal cells grown at variousconcentrations of the purine derivative AIT-034;

[0046]FIG. 17 is a graphical comparison of neurotogenic response ofneuronal cells grown at various concentrations of guanosine triphosphateand adenosine triphosphate with and without nerve growth factor;

[0047]FIG. 18 is a graphical comparison of nerve growth factor mediatedneurotogenic response to monophosphate, diphosphate, and triphosphatepurine derivatives of guanosine and adenosine;

[0048]FIG. 19 is a graphical comparison of cyclic GMP produced inneuronal cells grown in the presence of different concentrations of thepurine derivative guanosine;

[0049]FIGS. 20A, 20B and 20C are graphical comparisons of nerve growthfactor mediated neurotogenic responses of cells grown with and withoutthe purine derivative guanosine in the presence of variousconcentrations of three different inhibitors; FIG. 20A illustrates theneurotogenic response of cells grown in the presence of methylene blue,a guanylyl cyclase inhibitor, FIG. 20B illustrates the neurotogenicresponse of cells grown in the presence of various concentrations ofLY83583, also an inhibitor of guanylyl cyclase, FIG. 20C illustrates theneurotogenic response of cells grown in the presence of variousconcentrations of atrial natriuretic factor, a hormone which interactswith guanylyl cyclase;

[0050]FIG. 21 is a graphical representation of nerve growthfactor-mediated neurotogenic responses for neurons grown in the presenceof sodium nitrate, an inorganic nitric oxide donor;

[0051]FIGS. 22A and 22B are graphical comparisons of nerve growth factormediated neurotogenic response of neurons grown in the presence ofnitric oxide donors and scavengers of nitric oxide and carbon monoxide;FIG. 22A shows the neurotogenic response of cells grown in the presenceof various combinations of nitric oxide donors and hemoglobin and FIG.22B shows the neurotogenic response of cells grown in the presence ofvarious combinations of nitric oxide donors and methemoglobin;

[0052]FIG. 23 is a graphical comparison showing the nerve growth factormediated neurotogenic response of cells grown in various concentrationsof hemoglobin with or without the purine derivative guanosine;

[0053]FIG. 24 is a graphical comparison showing the nerve growth factormediated neurotogenic response of cells grown in various concentrationsof L-nitro arginine methylester (L-NAME) with and without the purinederivative guanosine;

[0054]FIG. 25 is a graphical comparison of the nerve growth factormediated neurotogenic response for cells grown in the presence ofvarious concentrations of zinc protoporphyrin IX (ZnPP), an inhibitor ofCO synthesis, with and without guanosine;

[0055]FIG. 26 is a negative control for the graphical comparison shownin FIG. 25 and is a graphical comparison of nerve growth factor mediatedneurotogenic response for cells grown in various concentrations ofcopper protoporphyrin IX (CUPP), with and without the purine derivativeguanosine; and

[0056]FIG. 27 is a graphical representation of the nerve growth factormediated neurotogenic response for neuron cells grown in the presence ofvarious concentrations of the purine derivative inosine pranobex.

DETAILED DESCRIPTION

[0057] In a broad aspect, the present invention is directed to methodsand associated compositions and medicaments for use in uniquely treatingmammalian cells and neurons to modify cellular or neural activity. Morespecifically, the present invention is directed to the use of effectivepurine derivatives to modulate the carbon dioxide dependent guanylylcyclase regulatory system within cells or neurons to produce a varietyof beneficial results, including the inducement of in vivo geneticexpression of naturally occurring neurotrophic factors and the resultantdirect administration of such naturally occurring genetically encodedmolecules to a mammal, the endogenous production of antioxidantcompounds and carbon monoxide, and the resultant ability to reducemammalian blood pressure. Preventing degenerative cellular destructionand treating disease conditions associated with cellular damage due tooxidative stress by free radicals also can be achieved with the methodsand compositions of the present invention.

[0058] In exemplary embodiments illustrative of the teachings of thepresent invention, particular purine derivatives were used to inducegenetic expression of encoded molecules, to stimulate neuritogenesis, toenhance neuronal growth and to modify the membrane potential of neuronsto produce increased learning capabilities in mammals. Exemplary studiesand treatments were performed as discussed below using various dosagesand routes of administration of selected exemplary purine derivativesrepresentative of compositions that are effective with the methods ofthe present invention. Of course, those skilled in the art willrecognize that the present invention is not specifically limited to theparticular compositions, dosages or routes of administration detailedbelow.

[0059] Depending upon the particular needs of the individual subjectinvolved, the compositions used may be administered as medicaments invarious doses and regimens to provide effective treatment concentrationsbased upon the teachings of the present invention. What constitutes aneffective amount of the selected composition will vary based upon suchfactors including the activity of the selected purine derivative, thephysiological characteristics of the subject, the extent and nature ofthe subject's neurodegradation or disorder and the method ofadministration. Exemplary treatment concentrations which have proveneffective in modifying neural activity range from less than 1 μM toconcentrations of 500 mM or more. Generally, initial doses will bemodified to determine the optimum dosage for treatment of the particularmammalian subject. The compositions may be administered using a numberof different routes including orally, topically, transdermally,intraperitoneal injection or intravenous injection directly into theblood stream. Of course, effective amounts of the purine derivatives mayalso be administered through injection into the cerebrospinal fluid orinfusion directly into the brain, if desired.

[0060] The methods of the present invention may be effected using purinederivatives administered as medicaments to a mammalian subject eitheralone or in combination as a pharmaceutical formulation. Further, thepurine derivatives may be combined with pharmaceutically acceptableexcipients and carrier materials such as inert solid diluents, aqueoussolutions or non-toxic organic solvents. If desired, thesepharmaceutical formulations may also contain preservatives andstabilizing agents and the like.

[0061] The methods and medicaments of the present invention provide forthe controlled long term modification of various types of cellular orneural activities including the in vivo production of naturallyoccurring genetically encoded molecules such as heme oxygenase andneurotrophic growth factors (including neurotrophins, pleiotrophins andcytokines), the direct administration of such in vivo producedmolecules, enhancing the effects of these molecules and neurotrophicfactors, and the stimulation of cell growth, function, protection, anddevelopment. Further, the present invention may be used to promoteneuritogenesis, to form collateral nerve circuits, to enhance theproduction of cyclic purine nucleotides, to enhance synapse formationand to alter the membrane potential of the neuron. These effects may beextremely beneficial in treating neurodegeneration and in increasinglearning capacity. similarly, inducing the in vivo production ofnaturally occurring endogenous antioxidant compounds may be extremelyuseful in treating and preventing disease conditions associated withoxidative damage including cancer, aging, and a variety of neurologicaldisorders.

[0062] For obvious practical and moral reasons, initial work in humansto determine the efficacy of experimental compositions and methods withregard to such afflictions is unfeasible. Accordingly, in the earlydevelopment of any drug or therapy it is standard procedure to employappropriate animal models for reasons of safety and expense. The successof implementing laboratory animal models is predicated on theunderstanding that the cellular or neurophysiology of mammals issimilar. Thus, a cellular or neurotropic response in a member of onespecies, for example, a rodent, frequently corresponds to the samereaction in a member of a different species, such as a human. Only afterthe appropriate animal models are sufficiently developed will clinicaltrials in humans be carried out to further demonstrate the safety andefficacy of a therapeutic agent in man.

[0063] With regard to neurodegenerative diseases and disorders and totheir clinical effects, the mouse model closely resembles the humanpathology of these conditions in many respects. Accordingly, it is wellunderstood by those skilled in the art that it is appropriate toextrapolate the mouse or “murine” model to humans and to other mammals.As with humans, mice are susceptible to learning disorders resultingfrom neuronal degradation, whether due to traumatic injury, oxidativedamage, age, disease or harmful chemical agents. Just as significantly,neurotropic factors appear to act in substantially the same manner in amurine model as they do in humans with remarkably similar neuronalreactions. Accordingly, for purposes of explanation only and not forpurposes of limitation, the present invention will be primarilydemonstrated in the exemplary context of mice as the mammalian subject.Those skilled in the art will appreciate that the present invention maybe practiced with other mammalian subjects, including humans, as well.

[0064] As will be shown by the data herein, several purine derivativeshave been found to work effectively in accordance with the teachings ofthe present invention. In particular, the data shows that guanosineappears to work well in stimulating the production of neurotrophicfactors and enhancing neuritogenesis. Similarly another exemplary purinederivative,4-[3-(1,6-dihydro-6-oxo-9-purin-9-yl)-1-oxopropyl]amino]benzoic acid(AIT-082) has been shown to stimulate the in vivo activation orderepression of naturally occurring genes and the resultant productionof naturally occurring genetically encoded molecules such asneurotrophic factors. It also induces the endogenous production of hemeoxygenase which in turn induces the endogenous production of carbonmonoxide and bile pigment antioxidant compounds. AIT-082 also increasesneuritogenesis, enhances the effects of neurotrophic factors and altersthe membrane potential of neurons thereby facilitating long termpotentiation of the cells.

[0065] AIT-082 is disclosed in U.S. Pat. No. 5,091,432 issued Feb. 25,1992 to a co-inventor of the present application and incorporated hereinby reference. Yet another exemplary composition which has been shown tobe suitable for use in the present invention is inosine pranobex orisoprinosine. Inosine pranobex, a mixture of inosine and DIP-PacBa at a1:3 molar ratio was found to enhance neuritogenesis and the effects ofneurotrophic factors in vitro. The different embodiments of the presentinvention presented above demonstrate the applicability of using variouspurine derivatives to modify cellular and neural activity throughmodulating the carbon monoxide dependent guanylyl cyclase system.

[0066] Exemplary preferred embodiments of the present invention involvethe treatment of cells or neurons with AIT-082 or4-[3-(1,6-dihydro-6-oxo-9-purin-9-yl)-1-oxopropyl]amino]benzoic acid. Asdiscussed previously, AIT-082 is a unique derivative of the purinehypoxanthine containing a para-aminobenzoic acid moiety. It is rapidlyabsorbed after oral administration and, after crossing the blood brainbarrier, enters the brain unchanged. It may be detected at levels ashigh as 3.3 ng/mg brain tissue 30 minutes after oral administration.AIT-082 induces the in vivo genetic expression of naturally occurringgenetically encoded molecules including heme oxygenase and neurotrophicfactors. As a result, the present invention is able to induce the directself-administration of these compounds to the treated cells and tostimulate the associated metabolic pathways and resultant physiologicaleffects. It also stimulates neurite outgrowth from neuronal cells whenadded alone to the cultures as well as enhancing the neurotogeniceffects of neurotrophic factors such as nerve growth factor (NGF). Moreimportantly, AIT-082 enhances working memory in old, memory deficientmice after either intraperitoneal and oral administration.

[0067] The neurotogenic activity of AIT-082 is inhibited by hemoglobin,by Methylene Blue, and by ZnPP, all scavengers of CO, but not by CuPP orby other inhibitors of nitric oxide synthase. Screening tests for invitro activity at known neurotransmitter and neuromodulator receptorswere negative. ZnPP is also known as an inhibitor of heme oxygenase Iwhich identifies the site of action of this exemplary guanylyl cyclasemodulating purine derivative. Heme oxygenase is a known heat shock(stress) protein. Heat shock proteins have been demonstrated to regulatethe conformation, intracellular transport, and degradation ofintracellular proteins. They are also involved in maintaining cellularviability during stress. Some in the art believe that decreased levelsor functioning of heat shock (stress) proteins may be one of the factorsleading to increased deposition of abnormally folded proteins and celldeath in the brains of Alzheimer's patients. Thus, the ability ofAIT-082 to induce the in vivo production of heme oxygenase evidences theimpact of the present invention on a variety of protective cellularpathways, indicating the therapeutic applicability of the presentinvention to the treatment of heart attack and stroke in addition totreating other disease conditions.

[0068] A further understanding of the present invention will be providedto those skilled in the art from the following non-limiting exampleswhich illustrate exemplary protocols for the identification,characterization and use of purine derivatives in accordance with theteachings of the present invention.

EXAMPLE 1 Plasma Levels of AIT-082 in Mice

[0069] Adult C57BL/6 mice were administered 30 mg/kg of AIT-082 insaline i.p. The animals were sacrificed by decapitation at 30, 45, 60and 90 minutes after administration of AIT-082. Blood was collected inheparinized tubes, mixed and centrifuged at 2000 rpm for 15 minutes. Theplasma supernatant was removed and stored at −70° C. until analysis. Ahigh pressure liquid chromatography system was developed for theanalytical measurement of AIT-082 in plasma and brain tissue. The assaydeveloped was selective for AIT-082 in the presence of a number ofclosely related purine molecules. The sensitivity of the method was 0.1microgram of AIT-082 per ml of plasma and 0.1 microgram of AIT-082 permilligram of brain tissue (wet weight).

[0070] The results of these determinations are shown in Table A andgraphically represented in FIG. 1 where plasma levels of AIT-082 areprovided at 30, 45 and 60 minutes after administration of 30 mg/kg i.p.to C57BL/6 mice. From the data, it was estimated that the blood level ofAIT-082 reached its peak at approximately 45 minutes and a plasmaelimination half-time of approximately 12 minutes with the k_(e1)=3.45hr⁻¹. TABLE A Plasma Levels of AIT-082 Time Level (min) (μg/ml ± S.E. 1542 ± 6  30 108 ± 13  45 437 ± 131 60 86 ± 24 90 20 ± 12

EXAMPLE 2 AIT-082 Crosses the Blood Brain Barrier

[0071] Brain tissue was analyzed from two animals receiving 30 mg/kgi.p. of AIT-082 and sacrificed 30 minutes after drug administration. Thebrains were rapidly removed and chilled on ice. Brain tissue wasdissected into cortex and remainder of the brain. Brain tissue (approx.250-300 mg wet weight) was homogenized with 5.0 ml of saline using aBrinkman Polytron tissue grinder and stored at −70° C. until analysis.Brain homogenates were deproteinized by ultrafiltration through GelmanAcrodisc filters; first through a 1.2 micron filter and then through a0.2 micron filter. A 30 μl sample was injected into the HPLC foranalysis as above. A standard curve was prepared by the addition ofknown quantities of AIT-082 to brain homogenates from untreated animals.Analysis of the brain tissue indicated that AIT-082 was detected in boththe cortex sample and the remaining brain samples from both animals. Theresults are shown directly below in Table B. TABLE B Brain Tissue Levelsof AIT-082 Brain wt Level of AIT-082 Sample # Brain Region (mg) (ng/mgbrain tissue) S3 Cortex 181 2.8 S3 Remainder 153 3.3 S4 Cortex 146 3.4S4 Remainder 217 2.3

[0072] This demonstration of the presence of AIT-082 in the brain tissueafter 30 minutes is critical in that it indicates that AIT-082 crossesthe blood-brain barrier without degradation.

EXAMPLE 3 AIT-082 Interacts with the Cholinergis System

[0073] Because of the finding that there is a severe loss of cholinergicneurons in the hippocampus in Alzheimer's disease patients, there hasbeen considerable interest in the effect on memory of compounds whichalter the activity of this system. Support for the cholinergichypothesis of memory comes from studies using lesions or a stroke model.Lesions of the CA1 region of the hippocampus appear to specificallydisrupt working memory. In the stroke model, occlusion of the vertebraland carotid arteries (30 minutes) produces specific cell loss in the CA1region of the hippocampus and a loss of working memory. In these modelsin aged rats, physostigmine, a cholinesterase inhibitor, has been shownto improve memory. THA, another drug which increases cholinergicfunction, was shown to improve memory in aged monkeys. The observationthat AIT-082 improves memory in the same general manner as physostigmineand THA raises the question of whether AIT-082 might have some effect onthe cholinergic system.

[0074] To elucidate the mechanisms by which AIT-082 improves memory,attempts were made to block its actions by co-administration of theshort-acting cholinergic antagonist atropine to mice and subjecting themto simple learning tests. Atropine reportedly has the ability to blockthe effects of physostigmine and THA. Mice were injected with AIT-082(30 mg/kg) 2 hr prior to testing on days 1 through 4. Atropine (0.5mg/kg) (28), was injected ½ hour prior to testing or 1.5 hours afterAIT-082 on day 3 only. All injections were i.p. After a reference run todetermine where the reward was placed in a T-maze, the mice wereretested to determine if they could remember the location of the reward.The percentage of correct responses is graphically represented in FIG.2.

[0075]FIG. 2 demonstrates that atropine blocked the memory enhancingactivity of AIT-082 on day 3 and that the effect was transient since theenhancing effects of AIT-082 reappeared on day 4 when no atropine wasadministered. This observation suggests that a cholinergic mechanism maybe involved in the action of AIT-082.

EXAMPLE 4 Effect of AIT-082 onN Acetylcholine Receptors

[0076] The interaction of AIT-082 with acetylcholine receptors wasdetermined by interference with the binding of QNB (3-quinuclidinylbenzilate) in mouse tissue using the method of Fields [J.Biol. Chem.253(9): 3251-3258, 1978]. There was no effect of AIT-082 in this assay.

[0077] In the study, mice were treated with AIT-082 at 30 mg/kg 2 hoursprior to sacrifice, decapitated and the tissue processed to obtainmembranes containing the acetylcholine receptors. When these tissueswere assayed in vitro, there was no effect of AIT-082 on affinity (Kd)for QNB when AIT-082 was administered under the same conditions asutilized in testing for effects on memory. There was a change in thenumber of receptors (B max) in cortex and striatum, with the cortexshowing a decrease and the striatum an increase in acetylcholine bindingsites. These data are consistent with the hypothesis that there is anincrease input to the cortex as a result of AIT-082 being administeredto the animals. Typically, an increased input will result in downregulation of the receptors.

EXAMPLE 5 Effect of AIT-082 on Receptor Ligand Binding in Vitro

[0078] AIT-082 was evaluated for its ability to inhibit ligand bindingto 38 isolated receptors. The receptors screened and their ligands were:

[0079] Adenosine

[0080] Amino Acids:

[0081] Excitatory Amino Acids (glycine, kainate, MK-801, NMDA, PCP,quisqualate and sigma);

[0082] Inhibitory Amino Acids (benzodiazepine, GABA-A, GABA-B, andglycine)

[0083] Biogenic Amines (dopamine-1, dopamine-2, serotonin-1,serotonin-2)

[0084] Calcium Channel Proteins (nifedipine, omegaconotoxin, chloride,potassium)

[0085] Peptide Factors (ANF, EGF, NGF)

[0086] Peptides: (angiotensin, arg-vasopressin-V1 and V2, bombesin, CCKcentral and peripheral, neurotensin, NPY, somatostatin, substance K,substance P, VIP)

[0087] Second Messenger Systems:

[0088] Adenyl Cyclase

[0089] Protein Kinase (phorbol ester and inositol triphosphate)

[0090] The testing was conducted under contract at Nova Labs (Baltimore,Md.). AIT-082 had no activity in any of the in vitro assays conducted.

[0091] Accordingly, while AIT-082 acts through the cholinergic nervoussystem (atropine blocks its activity), AIT-082 appears to act through amechanism that does not involve direct interaction with acetylcholinereceptors. It is of importance to note that in vitro, AIT-082 does notbind to the adenosine receptor.

[0092] AIT-082 was evaluated in a series of psychopharmacological teststhat were established in order to more fully evaluate the scope of itscentral nervous system activity. Among the tests utilized were:

[0093] (a) motor coordination, by the accelerating Rota-Rod treadmill,

[0094] (b) exploratory and home cage locomotor activity, by theStoelting activity monitor,

[0095] (c) anxiolytic activity, by the elevated Plus maze, and

[0096] (d) nocioception.

[0097] AIT-082 was compared with standard reference drugs.

EXAMPLE 6 AIT-082 Increases Motor Coordination in Mice

[0098] Motor coordination was measured using an accelerating Rota-Rodtreadmill for mice (Ugo Basile Co.). At various times after treatmentwith saline or drug, mice were placed on the Rota-Rod, which acceleratesto maximum speed over a 5 minute period. The time in seconds at whichthe subject falls off was recorded in Table C directly below. Eachanimal was tested 3 times and the mean time was recorded. TABLE C Effectof AIT-082 on Roto-rod performance AIT dose Time (mg/kg) (sec) Control 123 ± 64 0.005  162 ± 93 0.05 207* ± 73 0.5 184* ± 76 30.0 187* ± 6860.0 229* ± 80

[0099] Subjects receiving AIT-082 showed improved motor coordination byremaining on the roto-rod for longer periods of time when compared tocontrol (saline) or low doses (0.005 mg/kg).

EXAMPLE 7 AIT-082 Does not Inhibit Exploratory Activity

[0100] To measure exploratory behavior, subjects received saline orAIT-082 administration, were placed in a novel large cage (25×48×16 cm,W×L×H), and movement was measured at one-minute intervals for 30minutes. The large cage (San Diego Instruments, San Diego, Calif.) wasequipped with vertical detectors and rearing movements were alsorecorded. No effects were noted with respect to exploratory activityindicating that the subjects were not incapacitated.

EXAMPLE 8 AIT-082 Does not Inhibit Locomotor Activity

[0101] To measure home cage locomotor activity, the home cage was placedon a platform of an activity monitor (Stoelting Instruments). Home cagelocomotor activity movements were recorded at one minute intervals for15 minutes. Subjects received saline or AIT-082 and were returned totheir home cages. Ten minutes after injection, the home cage wasreplaced on the platform of the activity monitor. Home cage locomotoractivity movements were recorded at one minute intervals for 30 minutes.During the first five minutes, grooming activity was also monitored andrecorded. The results are shown in Table D directly below. TABLE DEffect of AIT-082 on locomotor activity Movements AIT dose (mean ± S.D.)(mg/kg) Pre-drug Post-drug Difference Control 1633 ± 434 1385 ± 492 248± 492 0.005 1884 ± 230 1375 ± 563 509 ± 429 0.05 1718 ± 606 1508 ± 456209 ± 340 0.5 1610 ± 349 1320 ± 689 290 ± 435 30.0 1440 ± 264 1098 ± 189342 ± 267 60.0 1690 ± 223 634* ± 223 1056* ± 154 

[0102] As shown by the data in Table D, at the high dose (60 mg/kg),subjects may have become more habituated to their environment andexhibited less movement after treatment with AIT-082. Otherwise, noeffects were noted.

EXAMPLE 9 AIT-082 Does not Substantially Increase Anxiety

[0103] A Plus maze was constructed of black plexiglass consisting of twoopposite-facing open arms (30×5 cm, L×W) and two opposite facing closedarms (30×5×15 cm, L×W×H). The walls of the closed arms were clearplexiglass and the four arms were connected by a central area 5×5 cm.The entire Plus maze was mounted on a base 38 cm above the floor.Testing consisted of placing the subject at one end of one of the openarms. The time the subject took to leave the start position (the first10 cm of the open arm) was recorded. The time it took for the subject toenter halfway into one of the closed arms was also recorded. When thesubject arrived at the half-way point in the closed arm, thethree-minute test session began. During the three-minute test session,the number of times the subject entered the open arms was recorded. Anentry was defined as placing at least two paws onto the platform of theopen arm. There was a slight anxiogenic effect of AIT-082 at 30 mg/kg,but this was not observed at a higher dose (60 mg/kg) or at the lowerdoses (0.005 to 0.5 mg/kg).

EXAMPLE 10 AIT-082 Does not Efffect Nocioception

[0104] Mice were placed on an electric hot plate (Omnitech) at 55° C.and the latency time until the subject licked his hind paw was measured.If there was no response by 45 seconds, the trial was terminated. Bythis test there was no effect of AIT-082 on nocioception.

EXAMPLE 11 AIT-082 Is not Toxic

[0105] Preliminary acute toxicity tests in rats and mice of AIT-082 havedemonstrated that the LD₅₀ is in excess of 3000 mg/kg when administeredby the oral or intraperitoneal route. AIT-082 has been evaluated underPanlabs's General Pharmacology Screening Program (Panlabs, 11804 NorthCreek Parkway South, Bothwell, Wash. 98011) and the results indicated anabsence of any toxicity when measured in their standard profile of 79different test systems.

[0106] By the nature of the chemical structure of AIT-082, it is notanticipated that the compound will be metabolized into any toxicmetabolites.

[0107] In conclusion, there were few deleterious effects of AIT-082 on avariety of psychopharmacological tests except for a slight anxiogeniceffect at one dose. There was an increase in motor coordination(roto-rod test) over a range of doses (0.05 to 60 mg/kg) and possibly alearning or habituation effect at one dosage (60 mg/kg) in the locomotortest.

[0108] Following psychopharmacological characterization of thisexemplary compound, further studies were conducted to demonstrate theneurogenic effects of the present invention.

EXAMPLE 12 AIT-082 Promotes Neuritogenesis in PC12 Cells

[0109] Much of the work performed in the characterization of thecompounds of the present invention involved the use of PC12 cells. Thesecells are derived from a rat pheochromocytoma and when grown in thepresence of NGF, extend neurites, cease cell division and assume manycharacteristics of sympathetic neurons. When cultured in the absence ofnerve growth factor (NGF), few PC12 cells have neurites greater than onecell diameter. Addition of saturating concentrations of NGF for 48 hoursstimulates neurite outgrowth in about 20-35% of the cells. Because theyconstitute a homogeneous population of neuronal-like cells, withoutcontaminating astroglia type cells, it is possible to study the directeffects of the purine based compounds on neurite outgrowth in thesecells.

[0110] To demonstrate neuronal modification by the exemplary compoundsof the present invention, a dose response curve of AIT-082 was generatedmeasuring the stimulation of neuritogenesis in PC12 cells. Cellscultured in RPMI 1640 with 1.5% horse serum and 1.5% fetal bovine serumwere replated onto poly-ornithine coated 24-well culture plates (2.5×10⁴cells per well). AIT-082 and NGF were added to the various culturesimmediately upon plating. After 48 hours, medium was removed and thecells immediately fixed in 10% formalin and PBS for 10 minutes. Cellsand neurites were counted within 2 days of fixation.

[0111] A neurite was defined as a process extending from the cell atleast 1 cell body diameter in length and displaying a growth cone at itstip. For each treatment, 2 representative microscope fields were countedfrom each of 6 sister cultures receiving identical treatments. The totalnumber of cells counted per well (approximately 100 cells) and the totalnumber of cells containing neurites in each well were used to determinefraction of neurite-bearing cells. The mean values (±SEM) were thendetermined for each of the treatments. To facilitate comparison neuriteoutgrowth was expressed relative to the proportion of cells bearingneurites in the presence of NGF alone (NGF=100%). The effects ofcompounds with and without NGF were compared by analysis of variance(ANOVA) followed by Tukey's test for significance.

[0112] The results are shown in FIG. 3 where the curve representsdifferent levels of AIT-082 plus saturating concentrations (40 ng/ml)2.5 S NGF. The center horizontal line represents control values forcells cultured in the presence of 40 ng/ml NGF alone. Upper and lowerhorizontal lines are indicative of confidence limits of NGF alone asdetermined using standard statistical methods.

[0113] As shown in FIG. 3, AIT-082 stimulates neuritogenesis andenhances NGF-stimulated neuritogenesis in PC12 cells at lowconcentrations (1 μM). Analysis of the data shows that AIT-082 was aseffective as NGF in promoting neuritogenesis in PC12 cells and enhancedthe optimal effects of NGF by 30%. For the purposes of comparison, andas will be discussed in more detail below, inosine and hypoxanthine areweakly effective in stimulating neuritogenesis and in enhancingNGF-stimulated neuritogenesis in PC12 cells but are effective at lowerconcentrations of 30-300 nM. Guanosine produces a significant effectsimilar to AIT-082 but at a higher concentration of 30-300 μM.

EXAMPLE 13 Effect of Inhibitors on AIT-082 Neuritogenesis

[0114] Age-related memory loss has been associated with loss ofNGF-dependent basal forebrain neurons. It can be ameliorated by i.c.v.infusion of NGF. The effect of AIT-082 on neuritogenesis alone and withNGF were studied using the protocol of Example 12. In order to study themechanism by which AIT-082 exerts its effects, a series of experimentswere conducted in which inhibitors were utilized to block or modifyspecific biochemical processes. All of the cultures contained NGF atoptimal dose (40 ng/ml) so the series without AIT-082 added representedthe effect of the inhibitors on NGF activity. Where indicated, AIT-082was added at 10 μM, its apparent, presently understood, optimal dose.Three selective inhibitors were used.

[0115] The results of these studies are shown below in Table E below,and FIGS. 4A, 4B, and 4C graphically present the proportion of cellsbearing neurites after 48 hours culture under the conditions indicated.The base line value was cells grown without NGF or AIT-082. TABLE EEffect of AIT-082 and selective inhibitors on neuritogenesis alone andwith NGF Con- AIT-082 AIT-082 + Inhibitor centration along¹ NGF aloneNGF None  0.2 ± 0.02  0.2 ± 0.02 0.26 ± 0.01 Methemoglobin 0  0.2 ± 0.020.26 ± 0.01 1 μM  0.2 ± 0.02 0.17 ± 0.02 Methylene Blue 0  0.2 ± 0.020.26 ± 0.01 5 μM 0.24 ± 0.03 0.10 ± 0.01 Zn 0 0.20 ± 0.02 0.26 ± 0.01Protoporphyrin IX 1 μM 0.22 ± 0.03 0.13 ± 0.01

[0116] Methemoglobin (MHb) captures and removes nitric oxide (NO) andcarbon monoxide (CO) from the culture media. MHb had no effect on NGFactivity but inhibited the action of AIT-082, implying that either NO orCO is involved in the action of AIT-82.

[0117] Methylene blue (MB) inhibits soluble guanylyl cyclase, the enzymewhich produces cyclic GMP (cGMP) a intracellular substance which, aspreviously discussed, is involved in the second messenger system ofnerve impulse transmission. MB had no effect on NGF activity butinhibited the action of AIT-082, implying that guanylyl cyclase isinvolved in the mechanism of action of AIT-082.

[0118] Zinc protoporphyrin IX (ZnPP) is an inhibitor of heme oxygenaseII (HO) which in turn produces CO. ZnPP had no effect on NGF activitybut inhibited the action of AIT-082. This identifies a potential site ofaction of AIT-082 as involving the production of HO. It also identifiesthe resultant production of CO as part of the mechanism of action ofAIT-082. These results are indicative of a number of important aspectsand features of the present invention.

[0119] As previously discussed, Ho is a heat shock (stress) protein.These proteins are believed to be part of protective mechanismsnecessary to maintain cellular viability during stress (see, e.g.Georgopoulos, C. and W. J. Welch, Role of the major heat shock proteinsas molecular chaperones. Annu. Rev. Cell Biol., 1993. 9: p. 601-634). Inaddition to regulating the confirmation, intracellular transport, anddegradation of intracellular proteins, Ho is also strongly implicated inthe production of potent antioxidant compounds produced by thedegradation of heme through the enzymatic activity of HO. In mammaliantissue the sole source of the protective antioxidant bile pigmentsbiliverdin and bilirubin is heme degraded by HO. There is substantialevidence that these bile pigments play an important physiological rolein cellular antioxidant defense mechanisms (Stocker, P., et al.,Bilirubin is an antioxidant of possible physiological importance.Science, 1987. 235: p. 1043-1047). Moreover, HO is found in the brain,and its level is known to greatly increase after heat shock (Ewing, J.F., S. N. Haber, and M. D. Maines, Normal and heat-induced patterns ofexpression of heme oxygenase-1 (HSP32) in rat brain: hyperthermia causesrapid induction of mRNA and protein. J. Neurochem., 1992. 58: p.1140-1149).

[0120] Consistent with this understanding, a number of neurotrophinsexert their neuro-protective effects by stimulating endogenous defensesagainst oxidative stress and damage by free radicals (Williams, L. R.,Oxidative stress, age-related neurodegeneration, and the potential forneurotrophic therapy, in Cerebrovasc. Brain Metab. Rev. 1995, RavenPress, Ltd.: New York, N.Y. p. 55-73. Mattson, M. P., B. Cheng, and V.L. Smith-Swintosky, Mechanisms of neurotrophic factor protection againstcalcium and free radical-mediated excitotoxic injury: implications fortreating neurodegenerative disorders. Exp. Neurol., 1993. 124: p.89-95). Because cytotoxic free radicals are suspected in the etiology ofa variety of neurodegenerative diseases, it is reasonable to concludethat the present invention is able to stimulate the production oractivity of HO to produce protective antioxidant compounds whichfunction to prevent degenerative cell destruction by oxidative freeradicals through the neutralization or sequestration of these toxicoxidative compounds.

[0121] Currently, it is believed by many skilled in the art that ALS,Parkinson's disease, and Alzheimer's disease may result from aninability to protect against accumulated cellular damage be freeradicals. Some practitioners skilled in the art have experimented withthe treatment of ALS through the administration of neurotrophins(DiStefano, P. S., Neurotrophic factors in the treatment of motor neurondisease and trauma. Exp. Neurol., 1993. 124: p. 56-59. Thoenen, H., R.A. Hughes, and M. Sendtner, Trophic support of motorneurons:Physiological, pathophysiological, and therapeutic implications. Exp.Neurol., 1993. 124: p. 47-55). Because the present invention is able toendogenously produce these protective compounds it provides an effectivetreatment for these and other degenerative cellular conditions. Thisprotective ability is particularly important for the treatment ofneurons because, unlike most cells which possess a variety of protectivemechanisms, such as high levels of glutathione, neurons are deficient inthis antioxidant source.

[0122] The ability of the present invention to stimulate the activity orproduction of HO has at least one additional direct physiologicalbenefit in mammals. When heme is degraded by HO into the bile pigmentsbilirubin and biliverdin, Co is also produced. Thus, the ability of thepresent invention to stimulate HO production and activity also providesthe present invention with the ability to stimulate the in vivo cellularproduction of CO. CO is known as an activator of soluble guanylatecyclase and relaxes vascular smooth muscle via a cGMP-dependentmechanism (Graser, T., Y. P. Verdernikov, and D. S. Li, Study of themechanism of carbon monoxide induced endothelium-independent relaxationin porcine coronary artery and vein. Biomed. Biochim. Acta, 1990. 49: p.293-296. Morita, T., et al., Smooth muscle cell-derived carbon monoxideis a regulator of vascular cGMP. Proc. Natl. Acad. Sci. USA, 1995. 92:p. 1475-1479). Recently, it was demonstrated by others in the art thatwithdrawal of the blood pressure lowering effects of CO throughinhibition of HO resulted in an increase in mammalian blood pressure(Johnson, R. A., et al., A heme oxygenase product, presumably carbonmonoxide, mediates a vasodepressor function in rats. Hypertension, 1995.25: p. 166-169). Accordingly, it is reasonable to conclude that theability of the present invention to stimulate the production of CO invivo will reduce mammalian blood pressure in addition to providingincreased antioxidant protection. The ability of the present inventionto directly induce the in vivo production of naturally occurringcellular compounds producing these dramatic physiological effects isunprecedented in the art.

EXAMPLE 14 Effect of Nitric Oxide Inhibitors on AIT-082

[0123] Nitric oxide is produced by the action of the enzyme nitric oxidesynthetase (NOS). Two chemicals that have been shown to selectivelyinhibit NOS are N-nitro-L-arginine methyl ester (L-NAME) andN-nitro-L-arginine (NOLA). Different levels of these chemicals wereadministered simultaneously with AIT-082 and neuritogenesis in PC12 wasmeasured using the protocol of Example 12. The results for L-NAME arepresented in Table F while the results for NOLA are presented in TableG. Both tables are shown directly below with graphical representationsof the data presented in FIGS. 5A and 5B. TABLE F The effect of L-NAMEon neuritogenesis AIT- Concentration of L-NAME (μM) 082 None 0.1 1.010.0  0 0.246 ± 0.017 0.259 ± 0.027 0.257 ± 0.013 0.251 ± 0.013  100.254 ± 0.008 0.220 ± 0.010 0.302 ± 0.027 0.254 ± 0.018 μM 100 0.309 ±0.027 0.257 ± 0.016 0.232 ± 0.019 0.289 ± 0.006 μM

[0124] TABLE G The effect of NOLA on neuritogenesis AIT- Concentrationof NOLA (μM) 082 None 0.1 1.0 10.0  0 0.246 ± 0.017 0.259 ± 0.009 0.311± 0.016 0.305 ± 0.017  10 0.254 ± 0.008 0.277 ± 0.016 0.312 ± 0.0290.298 ± 0.019 μM 100 0.309 ± 0.027 0.279 ± 0.027 0.295 ± 0.028 0.310 ±0.023 μM

[0125] As shown by the data in Tables F and G, neither of theseinhibitors of NOS were active in blocking the effect of AIT-082 onneuritogenesis. These results indicate that NO was not involved in themechanism of action of AIT-082.

EXAMPLE 15 EFFECT OF AIT-082 ON cGMP LEVELS IN PC-12 CELLS

[0126] To demonstrate CO-dependent guanylyl cyclase modification, cyclicguanosine monophosphate (cGMP) levels in PC12 cells were measuredfollowing addition of AIT-82. Initially, PC-12 cells were primed with 40ng/ml NGF for 3 days in low serum medium (1.5% horse serum+5% fetal calfserum). Cells were seeded onto assay plates in low serum mediumcontaining 40 ng/ml NGF and incubated for 1 hour. The medium was changedto low arginine medium (80 μM) with no serum and NGF and papaverine (100μM) where indicated. Test compounds were added for the indicated timeand the reaction was stopped by adding 5% TCA containing 10,000 dpm of³H-cGMP. cGMP was assayed by the radioimmunoassay method of Maurice[Mol. Pharmacol. 37: 671-681, 1990]. TCA was purified by adding powderedcharcoal (5 g) and filtering the mixture through Whatman #1 paper. Thisremoved contaminants in the TCA that otherwise interfere with theradioimmunoassay (RIA) of CGMP.

[0127] It was necessary to purify the cGMP from cAMP and othercontaminants before radioimmunoassay since these other materials caninterfere with the assay. Briefly, the TCA solution was applied to Dowexcolumns (50W-8X, 200-400 mesh) and eluted. A neutral alumina column wasthen placed under each Dowex column. The cGMP was eluted from the Dowexcolumns into neutral alumina columns by adding 4 mL of 0.05M HCl to eachDowex column. The neutral alumina columns were then sequentially rinsedwith 2 ml of HCl, 4 mL water and finally with 0.2M sodium acetate (pH6.2). The cGMP collected for the RIA, eluted in 1 mL of sodium acetatewith a recovery between 50-65%. The cGMP was assayed using a Dupont RIAkit. The results are graphically presented in FIG. 6.

[0128] As shown in FIG. 6, the addition of AIT-082 increased theproduction of cGMP in PC12 cells indicating that AIT-082 acts bymodifying the activity of the carbon monoxide-dependent enzyme guanylylcyclase.

EXAMPLE 16 Effect of AIT-082 on Genetic Expression of Neurothrophin mRNA

[0129] To demonstrate that AIT-082 induced the in vivo geneticexpression and resultant cellular production of neurotrophins, naturallyoccurring, genetically encoded molecules, as well as enhancing theiractivity, the following experiment was performed. Induction ofneurotrophin mRNA was determined by northern blot analysis of astrocytescultured with AIT-082, NGF, or both. The cells were harvested and RNAextracted at 24 hours after treatment.

[0130] More particularly, astrocytes from the cerebral cortex of NIHSwiss mice (Harlan) were isolated. Briefly, newborn pups (0-24 hours)were decapitated. Their brains were removed under aseptic conditions andwere placed in modified Dulbecco's medium (DMEM) containing 20%heat-inactivated horse serum (Hyclone)—(“complete medium”). Theneopallium was then dissected from each cerebral hemisphere and mincedinto 1 mm cubes.

[0131] The astrocytes were then isolated by mechanical dissociation. Thecubes were vortexed at maximum speed for one minute. The cell suspensionwas then passed first through 75 mm Nitex then through 10 mm Nitex. Theresulting cell suspension was diluted in complete medium to a finalconcentration of one brain per 10 ml of complete medium. Ten millilitersof the diluted cell suspension, was added to each 100 mm Falcon tissueculture plate (Fisher). After 3 days the medium was replaced with 10 mlfresh complete medium and subsequently was replaced twice weekly withDMEM containing 10% heat inactivated horse serum—(“growth medium”).After two weeks in culture the astrocytes formed a confluent monolayer.

[0132] For RNA extraction, astrocytes were trypsinized. The astrocyteswere then replated onto 100 mm PORN coated plates at a cell density of10⁶ cells per plate (10 ml growth medium). After 2 hrs PBS, Guo, or GTPat 10 mM were added to the appropriate plates. Total RNA was harvestedfrom 1.5×10⁷ cells for each treatment, 4 and 24 hrs after treatmentusing TRIzol reagent and supplier protocol (GIBCO BRL/Life Technologies,Inc.). For slot blots, total RNA was bound to Hybond-N filters(Amersham/United States Biochemicals) as described in Transfer andImmobilization of Nucleic Acids and Proteins to S & S Solid Supports (Sand S protocols: Schleicher & Schuell, New Hampshire, USA). Northernblots were also performed using 10-20 mg total RNA from each sample.These were electrophoresed in 1% agarose gels containing formaldehydeand blotted onto Hybond-N filters according to S and S protocols.

[0133] The blots were probed with P³²-labelled cDNA (NGF, NT-3 and BDNFprobes) or oligonucleotide probe (FGF-2) by hybridization inPiperazine-N,N′-bis-[2-ethanesulfonic acid] (PIPES) buffer (50 mM PIPES,pH 6.8; 50 mM NaH₂PO₄; 0.1 M NaCl; 5% SDS and 1 mM EDTA) overnight at50° C. The blots were then washed twice with (2×SSC, 0.1% SDS) washbuffer at room temperature for 20 minutes each, and then with (0.1×SSC,0.1% SDS) wash buffer twice at 52° C. for 20 minutes each. 1×SSC is0.15M NaCl and 15 mM sodium citrate, pH 7.0. Damp membranes were wrappedin Saran wrap and autoradiography was performed using Hyperfilm-MP(Amersham/USB) and a cassette with intensifying screens. Variousconcentrations (0.25 to 4 mg of total RNA), as determined byspectrophotometry, of each sample were blotted and probed so thatquantification could be done after insuring a linear film response.Quantification was performed using MCID Image Analysis (St. Catherine's,Ontario, Canada).

[0134] To provide probes, a cDNA clone of the mouse NGF gene in theplasmid pGEM.NGF(+), and cDNA clones of human NT-3 and BDNF inBluescript were isolated. After isolation, the cDNA probes were labeledwith ³²P-dCTP (ICN Biomedicals Canada, Ltd.) using a Random Primed DNALabeling Kit (Boehringer Mannheim Biochemica) as described in the kit.

[0135] A 40-mer antisense oligonucleotide was synthesized (MOBIX,McMaster University) as a probe for FGF-2. This was complementary to the5′ end of mouse FGF-2 coding sequence on the mRNA. The oligo was 5′end-labeled using polynucleotide kinase, One-Phor-All buffer, and theprotocol supplied by Pharmacia Biotech Inc., and ATPgP³² (ICNBiomedicals Canada, Ltd.).

[0136] The results of the study for the production of four differentneurotrophic factors are shown below in Table H. TABLE H Northern Blotanalysis of neurotrophin mRNAs from Astrocytes Neurotrophin NGF AIT-082AIT-082 (100 mM) + mRNA Control 40 ng/ml 100 mM NGF (40 ng/ml) NGF − −++ + FGF-2 + − ++ + BDNF + + + + NT-3 − − ++ +

[0137] The results indicate that AIT-082 induced the expression of mRNAsfor several neurotrophic factors, including NGF. More importantly, thesedata clearly establish that AIT-082 selectively and controllably inducedthe in vivo genetic expression of at least one naturally occurringgenetically encoded molecule in a mammal treated in accordance with theteachings of the present invention. Administering this exemplary purinederivative selectively induced the expression of mRNA encoding three ofthe four identified neurotrophic factors, NGF, FGF-2, and NT-3, but didnot induce activation or derepression of the gene encoding for BDNFmRNA. This selective control coupled with the ease of administrationprovided by the compounds and methods o the present inventioneffectively overcomes the limitations of the prior art. Rather thanadministering these molecular compounds directly to cells throughcomplex and potentially dangerous techniques, the present invention isable to treat a mammalian patient utilizing traditional, noninvasivedrug delivery routes that induce the treated cells to express thegenetic material encoding the desired compounds resulting in theirdirect in vivo delivery and administration. Though potentially useful inconjunction with modified genes or other molecular biology techniques,with the present invention, genetic modification is unnecessary.

[0138] It has been shown previously that, within the hippocampus fromAlzheimer's patients, there is an altered program of gene expressionleading to aberrant levels of mRNA for neurotrophic factors. A number ofanimal and clinical studies have demonstrated that administration ofsingle neurotrophins are inadequate to treat neurodegenerative disease.Accordingly, the ability of the compounds of the present invention tostimulate the production of multiple neurotrophin mRNAs within cellssubstantially increases their potential as treatments for a variety ofneurodegenerative diseases by providing a method for the effectivedirect administration of these naturally occurring genetically encodedmolecules to a patient through the induction of their in vivo geneticexpression.

[0139] The preceding examples show that AIT-082 stimulatesneuritogenesis in vitro in PC12 cells alone and enhances the effect ofnerve growth factor (NGF). Further, the neurotogenic effect of AIT-082was reduced by methemoglobin (which captures and removes nitric oxideand carbon monoxide), methylene blue (which inhibits guanylyl cyclase),and by zinc protoporphyrin IX (an inhibitor of heme oxygenase, whichproduces carbon monoxide). The neurotogenic effect of AIT-082 wasunaffected by L-NAME or NOLA, inhibitors of NO production. In addition,AIT-082 stimulated the production of a number of different neurotrophicfactors as evidenced by increased mRNA levels of these factors inastrocytes after AIT-082 administration in vitro. Moreover, sinceAIT-082 is orally active and rapidly passes the blood-brain barrier asshown in Example 2, it has significant therapeutic potential as anNGF-mimetic agent in Alzheimer's disease and in other neurodegenerativeand cellular diseases.

[0140] In view of the previous results, studies were performed todemonstrate the effectiveness of using AIT-082 to treat exemplaryneurodegenerative diseases. Loss of memory represents the core symptomof Alzheimer's disease as it does in a number of other neuralafflictions. Specifically working (or episodic) memory is impaired inAlzheimer's disease, amnesia, aging and after hippocampal lesions inmonkeys. The effects of AIT-082 in ameliorating this memory loss wasused to demonstrate the efficacy of the compounds of the presentinvention with respect to the treatment of neurodegenerative diseases.

EXAMPLE 17 Comparison of Memory Trace in Different Mice Strains

[0141] The win-shift T-maze paradigm has been shown to specificallymodel working memory in rodents and is a widely accepted method. Therodent's natural behavior is to forage for food when hungry andtherefore it will not return to the same location after it has consumedany food that was present. This model was not designed to account forall of the vast data on memory. Data from hypoxia and ischemia studies,procedures which selectively damage CA1 hippocampal cells, producedeficits in working memory while other types of memory are not affected.This strongly suggest that there are several types of memories whichhave different anatomical sites and most likely different neurochemicalinputs. Accordingly, while the win-shift model may not account for allneurochemical inputs involved in working memory, the model does providea useful art accepted tool in designing pharmacological experiments toprovide information on the mechanism by which memory can be modified.

[0142] Male Swiss Webster mice six months (young adult) and elevenmonths (old) of age, obtained from the National Institute on Aging, weremaintained in individual cages, on a 22 hour light/dark cycle withcontinuous access to water. Food was limited so that the mice stabilizedat 80% of free feeding weight. Mice were weighed and handled daily forone week. The win-shift model was run as described in the literature andconsists of a T-maze in which the correct response alternates after eachcorrect trial. The interval between trials is varied and allows for thedetermination of the longest period between trials that a subject canremember the correct response on the previous trial. This allows themeasure of the duration of the memory trace. A score of 5 (5 correctresponses per 10 trials, 50% correct) is considered chance; that is, theanimal does not remember which box it selected for positive reward onthe previous trial. The reward goal box is alternated after each correcttrial. Ten trials per mouse are run each day. If the animal establishesa spatial learning set (right side only), they would return to the samegoal each trial and have a correct response rate of significantly lessthan 50% correct. The latency time to leave the start box is recorded asa measure of motivation, the running time (the time from leaving thestart box to reaching the goal box) is recorded as a measure ofperformance, and the number of correct responses as a measure of memory.

[0143] The data in Table I illustrate the effect of increasing theinter-trial interval in young adult mice without any drug treatment.TABLE I Effect of inter-trial interval in win-shift paradigm⁽¹⁾Inter-trial Interval (seconds) 30 60 90 120 150 Swiss Webster mice 7.5*7.5* 5.0 C57BL/6 mice 7.0* 7.4* 7.0* 7.8 5.6

[0144] From the data in Table I, it can be seen that Swiss Webster miceare capable of remembering the win-shift strategy when the inter-trialdelay interval is 30 or 60 seconds. Few mice with saline treatmentscored above chance (50%) with the 90-second inter-trial delay interval.These data indicate that the “memory trace” in these animals disappearsbetween 60 and 90 seconds All drug evaluation tests in normal adultSwiss Webster mice were conducted with the 90-second inter-trialinterval except where indicated otherwise. In C57BL/6 mice, the durationof the memory trace was 120 seconds.

EXAMPLE 18 Effect of AIT-082 on Memory Trace Duration

[0145] The activity of AIT-082 was compared with tacrine (THA) andphysostigmine (PHY), experimental anticholinesterase agents whichenhance memory in animals. The drugs were also evaluated for theireffects on locomotor activity. In the win-shift memory paradigm, AIT-082was evaluated for its ability to induce tolerance after 18 days of drugadministration. In addition AIT-082 was tested for its activity tomodify learning in a modified T-maze discrimination task.

[0146] The drugs used in this example are4-[[3-(1,6-dihydro-6-oxo-9-purin-9-yl)-1-oxopropyl]amino] benzoic acid(AIT-082), as an exemplary potassium salt, tacrine hydro-chloride(tetrahydroaminoacridine, THA, Sigma Chemical Co., St. Louis, Mo.), andphysostigmine, hemisulfate salt (PHY, Sigma Chemical Co., St. Louis,Mo.). The drugs were dissolved in saline and prepared fresh daily. Allinjections were made at a volume of 0.1 ml/10 grams body weight. Whentesting drug effects, intraperitoneal (i.p.) injections of AIT-082 orTHA were made one hour prior to the start of testing. Due to its shorterduration of action, PHY was injected 30 minutes prior to testing.Control subjects receive a similar injection of saline (vehicle).

[0147] To determine the duration of the memory trace, subjects wereadministered drug or saline and 30 minutes (PHY) or 1 hour (AIT-082 orTHA) later they were given a single reference run with the milk rewardin both goal boxes. After the indicated inter-trial delay, subjects werereturned to the start box and given the first test trial with the milkreward only in the goal box opposite to the one entered on the previouscorrect trial. The subjects were given 10 trials with the rewardalternating only after correct responses.

[0148] To determine if tolerance to the biological effects of AIT-082developed, drug or saline was administered daily for 18 days prior tothe testing in the standard win-shift paradigm.

[0149] Subjects were also trained in the same T-maze used for thewin-shift model discussed above. As in the win-shift method, subjectswere shaped and then given a single reference run in which reward wasavailable in both goal boxes. The subject was only allowed to consumethe milk reward in the goal box selected. On the next run, the rewardand thus the correct response was in the same goal box selected for thereference run and was not alternated. The subject was required to learnthat there was no shift in the goal box for the correct response. Thesubjects were given 10 trials per day and continued until the subjecthad at least 8 out of 10 correct responses on two consecutive days. Thenumber of days to reach this criteria of performance was recorded. Afterthe subject reached criteria, the goal box for the correct response wasreversed. The number of days taken to reach criteria on reversal wasrecorded.

[0150] The results of the T-maze learning task and win-shift memory testare presented in Table J directly below. TABLE J Effect of AIT-082, THAand PHY at 90-Second Inter-trial Interval in Swiss Webster Mice Type ofTest⁽¹⁾ Control THA AIT-082 PHY Dosage (mg/kg) Saline 1.25 0.5 30.00.125 Win-shift Memory Test Correct responses 4.6 7.1* 6.5* 8.2* 6.5(Correct responses/10 trials) Latency time (seconds) 2.68 8.22* 1.952.03 Running time (seconds) 1.95 3.65* 2.20 1.95 2.65 LocomotorActivity⁽²⁾ 343 671* 323 378 N/T T-maze Learning(days to reach criteria)Learning 3.6 N/T⁽³⁾ 3.0 3.3 N/T Reversal 4.2 N/T   3.78 3.5 N/TTolerance 4.9 N/T   N/T 7.6* N/T (Correct responses/10 trials)

[0151] As shown by the data in Table J, AIT-082 treatment resulted in anincreased number of correct responses (memory) compared to salinecontrol. While the effect was in the same range as with THA and PHY,both THA and PHY also increased latency time (prolonged the time toleave the start box, evidencing decreased motivation) and THA increasedspontaneous locomotor activity. AIT-082 had no effect on learning orreversal and no tolerance developed to the memory enhancing effect ofAIT-082 after 18 days of pre-treatment. Only AIT-082 enhanced memoryfunction without affecting learning, motivation, performance andlocomotor activity. Similar data have been observed with oraladministration of AIT-082.

EXAMPLE 19 Effect of AIT-082 Dosage on Memory Trace Duration

[0152] The dose response and duration of action of AIT-082 was studiedin young adult Swiss Webster mice. The results are presented as thepercent correct response over chance; chance being 50% correct. As shownin FIG. 7, AIT-082 is active in improving memory in normal adult SwissWebster mice over a dose range from 0.5 to 60 mg/kg, with the optimaleffect at 20 to 30 mg/kg. Further, as shown in FIG. 8, the onset ofaction is rapid (1 hour, data not shown) and lasts for more than sevendays after a single dose of 60 mg/kg. Those skilled in the art willappreciate that the extended duration of the drug's effects willsubstantially lower the frequency of administration providing benefitsin terms of patient compliance and cost.

EXAMPLE 20 Effect of AIT-082 on Memory Trace Duration in C57BL/6 Mice

[0153] Previous work has established that normal adult Swiss Webstermice have a memory trace duration of 60 seconds in the win-shiftparadigm which may be increased by the administration of AIT-082. Inorder to further demonstrate the applicability and operability of themethods and compositions of the present invention, an alternative strainof mice having a different duration of memory trace was administeredAIT-082, using the preceding protocol. The results are shown in Table Kdirectly below. TABLE K Duration of Memory Trace in C57BL/6 MiceTreatment Groups Control AIT-082 Physostigmine Inter- (Saline) (30mg/kg) (0.125 mg/kg) trial No. above No. above No. above intervalchance/ chance/ chance/ (sec) Total^(#) Correct⋄ Total^(#) Correct⋄Total^(#) Correct⋄ 30 3/5  70 ± 11** 60 3/5  70 ± 16** 90 4/5  70 ± 6**120 4/5  78 ± 16** 150 1/5 56 ± 10 180 2/7 58 ± 12 4/6   70 ± 15** 3/6 65 ± 16* 210 4/6   78 ± 15** 1/6 53 ± 9 240 0/6 50 ± 6 270 0/6 50 ± 6

[0154] Typically, in the win-shift foraging paradigm, C57BL/6 mice havea duration of memory trace of 120 seconds. As shown in Table K, at 30mg/kg i.p., AIT-082 prolonged the duration of the memory trace to over210 seconds. While physostigmine also prolonged the duration of thememory trace from 120 to 180 seconds in this model, it was not as activeas AIT-082.

EXAMPLE 21 Treatment of Age Induce Memory Disorders Using AIT-082

[0155] In light of the preceding results, studies were performed todemonstrate that AIT-082 improves memory in mammals with neuronaldisorders as well as in healthy subjects. Twelve-month old male SwissWebster mice were screened for performance in the win-shift foragingtest. Subjects were tested at various time delays, beginning at 10seconds and increasing the inter-trial time interval to 30, 60, 90 and120 seconds. The results for untreated mice are shown in Table Ldirectly below. TABLE L Age-induced Working Memory Deficits in SwissWebster Mice Duration of No. of % Degree of Memory Memory Trace Subjectsof Subjects Impairment less than 10 seconds 6 25% Severe 10 seconds 833    Moderate 30 seconds 10 42    Mild Total 24 100

[0156] The results in Table L demonstrate that individual subjects canbe classified by the degree of working memory impairment. Subjects withsevere impairment could not remember the correct response at 10 secondswhile subjects with mild deficit could remember the correct responsewith a 30 second inter-trial interval but not at 60 seconds. Subjectswith a moderate deficit could remember the correct response with a 10second inter-trial interval but not at 30 seconds. Thus, the win-shiftmodel can detect age-induced impairments in working memory. As will beappreciated by those skilled in the art, this observation is importantsince it provides the ability to use age-matched subjects with varyingdegrees of impairment for evaluation of potential therapeutic agents.

[0157] Following the establishment of a baseline, six subjects in eachof the three groups were treated with AIT-082 (30 mg/kg, one hour beforetesting) or physostigmine (0.125 mg/kg, 30 minutes before testing) usingthe win-shift foraging test. The results are presented in Table Mdirectly below and graphically represented in FIG. 9. TABLE M Effect ofAIT-082 and PHY on the duration of memory trace in Swiss Webster micewith age-induced deficits Inter-trial Degree of Interval Control AIT-082PHY Deficit (sec) (Saline) 30 mg/kg 0.125 mg/kg Mild 60 0/6  6/6*  5/6*90 4/6 3/6 120 2/6 2/6 150 1/6 2/6 180 1/6 1/6 210 0/6 0/6 Moderate 300/6  4/6* 1/6 60 2/6 0/6 90 0/6 Severe <10 0/6 0/6 0/6

[0158] Six subjects had a severe deficit with no memory trace, theycould not remember the task at 10 seconds. None of these subjects showedmemory restoration with either AIT-082 or PHY treatment. In the sixsubjects with a moderate memory deficit who had a duration of memorytrace of 10 seconds, AIT-082 increased the duration of the memory traceto greater than 30 seconds in 4 subjects (67% of the subjects) andincreased the memory trace to greater than 60 seconds in two subjects(50%). In the six subjects with a mild memory deficit who had a durationof memory trace of 30 seconds, AIT-082 increased the duration of thememory trace in 2 subjects to 60 seconds, in 2 subjects to 90 secondsand in one subject each to 120 and 180 seconds. PHY increased theduration of the memory trace from 10 seconds to 30 seconds in only oneanimal in the moderate deficit group. In the mild deficit group, PHYincreased the duration of the memory trace in 2 subjects to 60 seconds,in one subject to 90 seconds and in two subjects to at least 180seconds. Thus, AIT-082 is more active than physostigmine in the moderatedeficit group and at least as active in the mild deficit group.

EXAMPLE 22 Treatment of Age Deficit Memory Disorders Using AIT-082

[0159] Twelve-month old male C57BL/6 mice were screened for performancein the win-shift foraging test. Subjects were tested at variousinter-trial time intervals. Subjects who could not perform to criteria(>60% correct) at 10 seconds delay were classified as having a severedeficit. Subjects who performed to criteria at 10 seconds but not at 30seconds were classified as having a moderate degree of deficit andsubjects who performed to criteria at 30 seconds but not at 60 secondswere classified as having mild deficit. As in Example 21, subjects ineach group were treated with either AIT-082 or PHY to determine theextent to which the working memory trace was prolonged. The results arepresented in Table N directly below and graphically represented in FIG.10. TABLE N Effect of AIT-082 and PHY on the duration of memory trace inC57BL/6 mice with age-induced deficits Inter-trial Degree of IntervalControl AIT-082 PHY Deficit (sec) (Saline) 30 mg/kg 0.125 mg/kg Mild 600/6  4/4*  7/8* 90  2/4* 3/8 120 2/8 150 2/8 180 2/8 210 0/8 Moderate 106/6  6/6* 6/6 30 0/6 4/6 1/6 60 1/6 0/6 90 0/6 Severe <10 0/6 0/6 0/6

[0160] In the mild deficit group, AIT-082 prolonged the duration of thememory trace from 30 to 90 seconds, and from 10 to 30 seconds in themoderate deficit group. While PHY prolonged memory in the mild group, itwas ineffective in the moderate group. Therefore AIT-082 restoredworking memory deficits in both normal mice and mice with age inducedneuronal disorder for both Swiss Webster and C57BL/6 strains.Specifically, the results show that AIT-082 restores working memory inmice with mild and moderate memory deficits. Based on the other Examplespreviously provided it is reasonable to conclude that it accomplishesthis restoration by modifying the carbon monoxide dependent guanylylcyclase system.

EXAMPLE 23 Prophylaxis of Age Deficit Memory Disorders Using AIT-082

[0161] It has been observed that age-induced memory deficits typicallybegin to manifest themselves in mice between 14 and 16 months of age.Therefore, we began treating mice at 14 months of age with AIT-082 (30mg/kg/day) in their drinking water. The animals were measured monthlyfor their memory using the win-shift foraging tests previouslydescribed. The results are shown in FIG. 11 and show that theadministration of AIT-082 delayed the onset and severity of memorydeficits

EXAMPLE 24 Prophylaxis of Alchool-Induced Deficit Memory Disorders UsingAIT-82

[0162] In order to demonstrate the broad applicability of the presentinvention with respect to different types of neurodegenerativedisorders, AIT-082 was used to retard the production of alcohol inducedmemory deficit. Six month old male C57BL/6 mice were evaluated in thewin-shift model in combination with treatment with ethanol, anon-specific memory suppressant, and AIT-082. Subjects were treated withsaline (control) or AIT-082 (30 mg/kg. i.p.) 1 hour prior to testing.Ethanol was administered at a dose of 0.5 gm/kg i.p. ten minutes priorto testing. The results of a pilot study are presented in Table Odirectly below. TABLE O Working memory deficit produced by ethanol andits reversal by AIT-082 Treatment Ethanol + Control Ethanol AIT-082Correct trials^(1,2) 8.08 ± 0.29 6.5 ± 26* 7.89 ± 0.54 † Latencytime(sec)² 1.24 ± 0.17 1.18 ± 0.10 1.77 ± 0.27 Running time(sec)² 1.44 ±0.35 1.17 ± 0.08 3.22 ± 0.61*† Number of subjects 13 13 9

[0163] The results in Table O demonstrate that it is possible toidentify a dose of a blocking agent that can produce a memory deficit asmeasured in the win-shift model. Ethanol was selected as a non-specificblocking agent and its effects were reversed by administration ofAIT-082 prior to the treatment with ethanol. Therefore it would appearfeasible to evaluate other more specific blocking agents which haveactivity at specific receptor sites.

[0164] In addition to AIT-082 other purine derivatives are believed toplay a role in neuronal survival, synaptogenesis and recovery offunction following injury or cell death in the central nervous system.For example, similarities between guanosine and AIT-082 indicate thatAIT-082 and guanosine act through comparable mechanisms. That is, bothappear to act as carbon monoxide dependent guanylyl cyclase modulators.Further, it is known that after cells are damaged, they leak massiveamounts of both purine nucleosides and nucleotides to the extracellularspace. The extracellular concentration of guanosine in the region of afocal brain injury may reach 50 mM and is elevated up to five fold forat least seven days. Therefore, following injury, astrocytes or glia andneurons are exposed to high extracellular concentrations of guanosine.

[0165] Accordingly, the following studies were undertaken in order todemonstrate the effectiveness of using other exemplary purinederivatives such as guanosine to modulate the carbon monoxide dependentguanylyl cyclase system.

EXAMPLE 25 Astrocytes Produce Tropic Factors Upon Exposure to Guanosineand GTP

[0166] Astrocytes appear to proliferate in response to extracellularguanosine or guanosine triphosphate (GTP). GTP or guanosine may alsostimulate the release of trophic factors from cultures of neocorticalastrocytes from neonatal mouse brains. Astrocytes were incubated withdifferent concentrations of guanosine of GTP respectively. Neurotrophinimmunoreactivity in the culture medium from treated cells was thenmeasured by ELISA.

[0167] Briefly, 96 well Falcon plates (Fisher) were coated with 1 mg/mlof sheep mono-specific anti-NGF IgG (affinity column purified) containedin 0.1M sodium carbonate buffer pH 9.6. After an overnight incubation at4° C. blocking solution (PBS with 10% goat serum) was added to removeexcess antibody. After a four hour incubation at room temperature theplates were washed 3 with PBS containing 0.05% Tween 20. The conditionedmedia and standard 2.5S HPLC purified NGF were added and incubatedovernight. The next day plates were washed 3 times with PBS-0.05% Tween20. The secondary antibody, rabbit mono-specific anti-NGF IgG conjugatedwith b-galactosidase (Pierce-SPDP method) (1:500 dilution) was added.The plates were incubated overnight at 4° C. The next day the plateswere washed 3 times with PBS-0.05% Tween 20. To each well substrate, 0.2mM 4-methylumbelliferyl-b-galactoside (MUG) in 0.1M phosphate buffer (1mM MgCl₂ pH 7.2) was added. After a 4 hour incubation at roomtemperature the reaction was stopped by the addition of 0.1M glycine, pH10.3. Samples were then read using Microfluor ELISA reader (excitation360 nm; emission 450 nm). The sensitivity of this assay was 10 pg/wellNGF.

[0168] The ELISA assay detected neurotrophins NGF and NT-3 with almostequal affinity and BDNF with 100 times less affinity. As shown in FIGS.12A and 12B, both guanosine and GTP increased the amount of NGF-likeimmunoreactivity in the culture medium. The astrocytes exposed to thevarious levels of guanosine produced a much stronger response than thoseexposed to equivalent concentrations of GTP.

EXAMPLE 26 Astrocytes Produce Neurotrophic Factors Upon Exposure toGuanosine

[0169] In order to confirm the results of the previous assay, mRNAlevels of the tropic factors FGF-2 and NGF were measured in astrocyteswhich had been exposed to guanosine. The mRNA levels were measured usingthe same protocol used previously in Example 16. As shown in FIGS. 13Aand 13B, the addition of guanosine increased NGF and FGF-2 mRNA at 4hours and at 24 hours, respectively, after it was added to astrocytes.The observed increase in neurotrophin mRNA is important following braininjury or recovery from brain injury when the extracellularconcentration of guanosine is considerably high. As cells are exposed toa high concentration of guanosine for several days following braininjury, this data indicates that guanosine may be responsible for someof the recovery of function.

[0170] As previously discussed, an agent that can penetrate the bloodbrain barrier and increase concentrations of neurotrophic factors asmeasured here by mRNA levels should have a substantial positive effecton neuronal survival and on the formation of collateral nerve circuits.In turn, this should enhance functional recovery in many differentneurological diseases or after damage to the nervous system.

EXAMPLE 27 Neurons Undergo Neuritogenesis Upon Exposure to GUANOSINE

[0171] In addition to changes in glia or astrocytes, important neuronalchanges also take place following focal brain injury. Neuritic processesof surviving neurons may undergo neuritogenesis. Accordingly, based onprevious results using AIT-082, studies were performed to demonstratethat guanosine may also modify carbon monoxide guanylyl cyclase tostimulate neuritogenesis. As previously discussed, because PC12 cellsconstitute a homogeneous population of neuronal-like cells, withoutcontaminating astroglia-type cells, the direct effects of the exemplarypurine derivatives of the present invention on neurite outgrowth inthese cells can be observed easily. Accordingly, PC12 cells were exposedto guanosine and adenosine with and without NGF and monitored as inExample 12. The effects of exposure to purine derivatives with NGF areshown in FIG. 14A while exposure without NGF is shown in FIG. 14B. Adirect comparison of the effects of these purine derivatives with andwithout the presence of NGF is shown for each compound in FIG. 14C.

[0172] As shown in FIG. 14A, guanosine, but not adenosine, enhanced theneurite outgrowth induced by NGF in PC12 cells after 48 hours. Theenhancement was significant over that of NGF alone at guanosineconcentrations of 30 and 300 mM. Adenosine did not enhance NGF inducedneurite outgrowth at any concentration. This indicates that neuriteoutgrowth induced by purines is not just a generalized phenomenon.5′-N-ethylcarboxamidoadenosine (NECA), an adenosine A₁ and A₂receptoragonist, also enhanced neuritogenesis, but not to the same extent asguanosine.

[0173] On their own, in the absence of NGF, both adenosine and guanosineslightly increased the proportion of cells with neurites as shown inFIG. 14B. The effects of guanosine at both 30 and 300 mM was greaterthan adenosine at the same concentrations. In the presence of (NECA),there was little stimulation of neurite outgrowth. Because the effectsof the compounds in the presence of NGF were much more readily scoredand less variable from experiment to experiment than with the compoundsalone, most of the data for enhancement of neurite outgrowth wasdetermined in the presence of NGF.

[0174] The comparative data shown in FIGS. 14A and 14B and emphasized inFIG. 14C show that guanosine causes some neurite extension, but can alsoreact synergistically to enhance the trophic effects of NGF. Adenosine,although slightly enhancing neurite outgrowth on its own does notenhance the effects of NGF. Interestingly, NECA but not adenosine couldsynergistically enhance the actions of guanosine, both in the presenceand absence of NGF as shown in FIG. 14C. The fact that adenosine did notincrease NGF-dependent neurite outgrowth in PC12 cells but thatguanosine did, suggests that they interact differently with PC12 cells.Adenosine would interact with adenosine receptors, such as the A₂purinoceptor. This would activate adenylate cyclase which increasesintracellular cAMP. NECA apparently acts in this manner. But the effectsof NECA were synergistic with those of guanosine. This indicates thatguanosine and NECA use different signalling pathways to enhance neuriteoutgrowth.

EXAMPLE 28 Various Purine Derivatives Provide Different Rates ofNeuritogenesis

[0175] In view of the previous results, other exemplary purinederivatives were examined to demonstrate the specificity of thosecompounds which modulate carbon monoxide dependent guanylyl cyclase tomodify neural activity. Specifically, different concentrations of thepurine derivatives inosine, hypoxanthine and xanthine were tested in thepresence of NGF using the protocol of Example 12 to demonstrate theirability to modify neural activity.

[0176] As shown in FIG. 15A, inosine only slightly enhanced neuriteoutgrowth over that produced in cells treated with NGF alone. This wastrue for concentrations of inosine ranging from 0.3 to 300 mM. FIG. 15Aalso shows that the action of inosine on the enhancement of neuriteoutgrowth was much less effective than that of guanosine.

[0177]FIGS. 15B and 15C also show that hypoxanthine and xanthine eachproduced results similar to that of inosine on NGF-inducedneuritogenesis. In FIG. 15C xanthine, in concentrations from 0.3 to 30mM (300 mM was toxic to the cells), only slightly enhanced NGF-inducedneurite outgrowth. FIG. 15B shows that hypoxanthine showed the greatest,although still modest, enhancement at concentrations of 0.3 and 300 mM,although other concentrations had no significant enhancement. Eventhough some enhancement of neurite outgrowth was observed withhypoxanthine, the relative amount of enhancement was not nearly as greatas was the effect of guanosine. These results indicate that inosine,xanthine and hypoxanthine do not modulate the carbon monoxide-dependentguanylyl cyclase system to modify neural activity but rather influenceother signaling mechanisms.

EXAMPLE 29 Effects of AIT-34 on Neuritogenesis

[0178] To demonstrate the effects of compounds similar to AIT-082 onneuritogenesis, PC12 cells were exposed to AIT-34, otherwise known as3(1,6dihydro-6-oxo-9h purin-9-yl)-N-[3-(2-oxopyrrolidin-1-yl)propyl]propanamide, during growth and monitored according to Example 12.As shown in FIG. 16, different concentrations of AIT-034 did not enhanceNGF-induced neuritogenesis as is observed with AIT-082.

EXAMPLE 30 Effects of ATP and GTP on Neuritogenesis

[0179] To further demonstrate that purine derivatives having differentfunctional groups may be used in accordance with the teachings of thepresent invention, PC12 cells were exposed to adenosine triphosphate(ATP) and guanosine triphosphate (GTP) and monitored for neuritogenesisusing the protocol of Example 12.

[0180] In a manner very similar to the actions of adenosine andguanosine on neurite outgrowth in PC12 cells, their correspondingnucleotides ATP and GTP had parallel effects on neurite outgrowth. Asshown in FIG. 17, ATP did not enhance neuritogenesis in either NGFtreated cells or on its own. In sharp contrast, GTP at 30 and 300 mM,did enhance neuritogenesis in the presence of NGF and further elicitedneurite outgrowth on its own.

[0181] However, as shown in FIG. 18, GTP did not appear to be acting asa source from which guanosine was released in a controlled manner. IfGTP was hydrolyzed to guanosine diphosphate (GDP), guanosinemonophosphate (GMP) and finally to guanosine by ectoenzymes, one wouldpredict that GDP and GMP would also enhance neurite outgrowth from PC12cells. Yet, neither GDP nor GMP were effective alone or with NGF ineliciting neurite outgrowth. By way of comparison, the adenine-basedcompounds all had an inhibitory effect.

EXAMPLE 31 Guanosine But not GTP Increases cGMP in PC12 Cells

[0182] Based on the previous examples, a study was conducted todemonstrate the neurotogenic mechanisms of GTP and guanosinerespectively. Guanosine and GTP have been shown to increaseintracellular cyclic 3′,5′-guanosine monophosphate (cGMP) in arterialsmooth muscle. Since cGMP analogues have been reported to stimulateneurite outgrowth from neuroblastoma cells it was possible that bothguanosine and GTP might exert their effects through increasingintracellular cGMP. As shown in FIG. 19, guanosine increasedintracellular cGMP in PC12 cells as determined by radioimmunoassay usingthe protocol detailed in Example 15. Such an increase would be expectedof a carbon monoxide dependent guanylyl cyclase modulator. In contrast,it was found that GTP did not increase levels of cGMP, indicating thatany GTP-stimulated neuritogenesis occurs by another mechanism.

EXAMPLE 32 Use of Non-Selective Inhibitors of Guanylyl Cyclase ReducesGuanosine Neuritogenesis

[0183] To demonstrate that guanosine modifies the carbonmonoxide-dependent guanylyl cyclase system, studies were conducted toshow that increased levels of intracellular cGMP were necessary forguanosine to enhance NGF's neurotogenic effects on PC12 cells. Inparticular, different concentrations of three inhibitors of guanylylcyclase were added to PC12 cells with guanosine. Neuritogenesis was thendetermined using the protocol of Example 12.

[0184] Methylene Blue (MB) inhibits soluble guanylyl cyclase (sGC), theenzyme that synthesizes cGMP. As shown in FIG. 20A the addition of MB(0.1-5 mM) to cultures of PC12 cells abolished the synergistic effectsof guanosine with NGF. Conversely, MB had no effect on NGF-stimulatedneurite outgrowth.

[0185] LY83583 inhibits both particulate and sGC. FIG. 20B shows thatthe neurite outgrowth response elicited by guanosine was inhibited byLY83583, but the response elicited by NGF was unaffected. The mechanismby which LY83583 inhibits guanylyl cyclase is unresolved, but is likelyindirect, involving glutathione metabolism. Therefore, two non-selectiveinhibitors of guanylyl cyclase, each with a different mechanism ofaction, attenuated the neurotogenic action of guanosine.

[0186] These data indicate that guanosine and NGF act through differentmechanisms. They also indicate that increases in intracellular cGMP werenecessary, although possibly not sufficient, for guanosine to exert itsneurotogenic effects.

[0187] To test whether increases in cGMP were sufficient to causeneurite outgrowth, atrial natriuretic factor (ANF) was added to cellcultures in a manner similar to that used for guanosine. ANF is ahormone whose only known signal transduction pathway is throughactivation of particulate guanylyl cyclase. As shown in FIG. 20C, ANF,like guanosine, enhanced NGF-stimulated neurite outgrowth from PC12cells indicating that increased intracellular cGMP production, inducedby carbon monoxide dependent guanylyl cyclase or other mechanismsassisted in stimulating neurite outgrowth.

EXAMPLE 33 Nitric Oxide or Carbon Monoxide Promotes GuanosineNeuritogenesis

[0188] Because guanosine increased intracellular cGMP as shown inExample 31, studies were performed to demonstrate whether its signalcould be transduced through production of NO or CO. If NO was involved,then addition of nitric oxide donors that liberate NO should mimic theeffects of guanosine.

[0189] PC12 cells were grown for 48 hours in the presence of sodiumnitroprusside (SNP) or sodium nitrite (SN), both of which liberate NO.Alone, neither SNP nor SN elicited neurite outgrowth from PC12 cells.However, like guanosine, both SNP and SN enhanced NGF-mediated neuriteoutgrowth in a synergistic manner as shown for the addition of SN inFIG. 21. Further confirming the effect, FIGS. 22A and 22B show that theneurotogenic properties of the NO donors were inhibited by bothhemoglobin (Hb) and methemoglobin (MB). Both are substances whichscavenge NO and CO with high affinity and preclude these agents frombeing used as signal transmitters.

[0190] Accordingly, if NO or CO mediates the neurotogenic effects ofguanosine, then these effects should be reduced by addition ofhemoglobin to the cultures. The expected effect is clearly shown in FIG.23 where Hb (0.1-1 mM) inhibited the neurotogenic effects of guanosinebut not those of NGF. This indicates that the neurotogenic action ofguanosine, but not that of NGF, requires synthesis of NO or CO.

[0191] Several facts indicate that it is CO rather than NO whichinteracts with guanosine to modify neural activity. For example, if theeffects of guanosine were mediated through NO, then addition ofguanosine to the PC12 cells should stimulate cNOS in PC12 cells toproduce NO. However, cNOS had not been reported in PC12 cells anduntreated (guanosine and NGF naive) PC12 cells did not stain fordiaphorase, an enzyme that co-localizes with NOS. Since cNOS iscalcium/calmodulin-sensitive, its activity should increase after addinga calcium ionophore, thus leading to increased cGMP levels. Addition ofthe ionophore A23187 to cultures of PC12 cells failed to elicit anincrease in cGMP.

EXAMPLE 34 Carbon Monoxide, Not Nitric Oxide, Mediades the Effects ofGuanosine on Neuritogenesis

[0192] Based on the results of the previous examples, studies wereperformed to demonstrate that the purine derivatives of the presentinvention, including guanosine, modulate the carbon monoxide-dependentguanylyl cyclase system to modify neural activities.

[0193] As in Example 6 where it was shown that carbon monoxide mediatesthe effects of AIT-082 through the use of inhibitors, the sametechniques demonstrate that guanosine also interacts with the carbonmonoxide dependent system. Specifically, as shown in FIG. 24, the cNOSinhibitor L-nitro arginine methyl ester (L-NAME) did not affect theability of guanosine to enhance NGF-mediated neurite outgrowth. Thesedata confirm that cNOS was not involved in the signal transductionpathway that mediated the neurotogenic effects of guanosine on PC12cells.

[0194] To further demonstrate that CO, rather than NO, mediated theneurotogenic effects of guanosine, zinc protoporphyrin IX (ZnPP), whichinhibits heme oxygenase and hence inhibits CO synthesis, was added tothe cells during growth. As shown in FIG. 25, ZnPP abolished theneurotogenic effects of guanosine, but did not affect those of NGF. Incontrast, a related protoporphyrin derivative, copper protoporphyrin IX(CuPP), does not inhibit heme oxygenase. Accordingly, FIG. 26 shows thatcopper protoporphyrin IX did not reduce the ability of guanosine toenhance NGF-dependent neurite outgrowth from PC12 cells. As withAIT-082, these data indicate that guanosine increased CO synthesis. Inturn, CO activated sGC and increased intracellular GMP, therebypromoting neuritogenesis.

EXAMPLE 35 Inosine Pranobex Enhances Neuritogenesis

[0195] To provide further evidence of the scope and operability of thepresent invention, neurotogenic studies were performed using inosinepranobex. Specifically, inosine pranobex is a mixture of inosine andDIP-PacBa at a 1:3 molar ratio. Various concentrations of this compoundwere added to PC12 cells with NGF which were then monitored according tothe protocol of Example 12.

[0196] As shown in FIG. 27, inosine pranobex substantially enhanced theamount of neurite outgrowth of the treated cells. The curve shown inFIG. 27 represents the different levels of inosine pranobex plussaturating concentrations of NGF while the horizontal lines representthe NGF control with attendant confidence levels. Here the treated cellsare above the control baseline at most of the selected concentrations.

[0197] The modification of cellular and, more specifically, neuralactivity in accordance with the teachings of the present invention maybe used to treat a wide variety of cellular and neurodegenerativediseases in order to provide recovery of cellular or neural function.Thus, the present invention may be used to treat cellular andneurodegeneration from any cause including oxidative stress, disease,trauma, age, and exposure to harmful physical or chemical agents.Similarly, the methods and medicaments disclosed herein may be used totreat neurological diseases including, but not limited to, Alzheimer'sDisease and related degenerative disorders, Parkinson's disease andrelated disorders such as striatonigral degeneration, spino-cerebellaratrophies, motor neuronopathies or “motor system diseases” includingAmyotrophic Lateral Sclerosis, Werdnig-Hoffmann disease,Wohlfart-Kugelberg-Welander syndrome and hereditary spastic diplegia,damage to neurons by ischemia (as in strokes), anoxia, or hypoglycemia(as, for example, after prolonged circulatory arrest), Huntington'sdisease, cerebral palsy, multiple sclerosis, psychiatric disordersincluding affective disorders, schizophrenia, epilepsy and seizures,peripheral neuropathies from any cause, learning disabilities anddisorders of memory. Also, damage to neurons or their processes byphysical agents such as radiation or electrical currents or by chemicalagents including alcohol, aluminum, heavy metals, industrial toxins,natural toxins and legal or illegal drugs may be treated. The methodsmay further be used to treat victims of trauma to the brain or spinalcord resulting in neuronal damage or age related conditions such asbenign forgetfulness and deterioration of sensory, motor, reflex orcognitive abilities due to loss of neurons or neuronal connectivity.Simply administering an effective dosage of at least one of the carbonmonoxide dependent guanylyl cyclase modulating purine derivatives of thepresent invention to a subject suffering from any of the foregoingcellular or neural disorders will induce intracellular changes producingrestoration of function.

[0198] Specifically, modification of the carbon monoxide dependentguanylyl cyclase system in accordance with the teachings of the presentinvention produces changes in neural activity in neurons and glia cellsincluding astrocytes. For example, using the present invention theneural activity of astrocytes may be modified to synthesize variousneurotrophic factors and cytokines including fibroblast growth factor(FGF), nerve growth factor (NGF), brain derived neurotrophic factor(BDNF) and neurotrophin-3 (NT-3). These factors can influence thesprouting of neuritic processes from surviving neurons as well aspromote the development of new cells. New synapses may then form andprovide some recovery of function. These neurotrophic factors also playa neuroprotective role. Thus, inducing their production can amelioratefurther neural damage.

[0199] Numerous purine derivatives may be used in accordance with theteachings of the present invention. However, the ability to modifyneural activity by modulating the carbon monoxide dependent guanylylcyclase system is not a general property of all purines or purinederivatives. For example, as shown in the data below, inosine,adenosine, hypoxanthine and xanthine were all relatively ineffective atmodifying neural activity. Other purine derivatives which failed tomodify neural activity include 3-(6-amino-9H-purin-9-yl)propionic acid,ethyl ester (AIT-0026),3-(1,6-dihydro-6-oxo-9H-purin-9-yl)-N-{3-(2-oxopyrolidin-1-yl)propyl]propanamide(AIT-0034) and propentofylline. Moreover, while other purines and purinederivatives such as 5′-N-ethylcarboxamidoadenosine (NECA) were shown tostimulate neurite outgrowth, they did not do so by modulation of thecarbon monoxide dependent guanylyl cyclase mechanism. Accordingly, thescope of the invention is defined by the functional reactivity of purinederivatives which modify cellular or neural activity as described hereinand as shown by the data presented. Of course, those skilled in the artwill appreciate that functionally equivalent isomers, analogs andhomologs of the compounds of the present invention may be substituted.

[0200] Those skilled in the art will further appreciate that the presentinvention may be embodied in other specific forms without departing fromthe spirit or central attributes thereof. In that the foregoingdescription of the present invention discloses only exemplaryembodiments thereof, it is to be understood that other variations arecontemplated as being within the scope of the present invention.Accordingly, the present invention is not limited to the particularembodiments which have been described in detail herein. Rather,reference should be made to the appended claims as indicative of thescope and content of the present invention.

What is claimed is:
 1. A method for the treatment of mammalian diseaseconditions associated with cellular damage due to oxidative stress, saidmethod comprising the step of inducing the in vivo production of atleast one naturally occurring endogenous antioxidant by treating amammalian subject with an effective amount of at least one carbonmonoxide dependent guanylyl cyclase modulating purine derivative.
 2. Themethod of claim 1 wherein said carbon monoxide dependent guanylylcyclase modulating purine derivative is selected from the groupconsisting of guanosine,4-[[3-(1,6-dihydro-6-oxo-9H-purin-9-yl)-1-oxo-propyl]amino]benzoic acid,and inosine pranobex.
 3. The method of claim 1 wherein said effectiveamount of said at least one carbon monoxide dependent guanylyl cyclasemodulating purine derivative produces a treating concentration of atleast 1 μM.
 4. The method of claim 2 wherein said mammalian subject istreated by orally administering said at least one carbon monoxidedependent guanylyl cyclase modulating purine derivative.
 5. The methodof claim 2 wherein said mammalian subject is treated by injecting saidat least one carbon monoxide dependent guanylyl cyclase modulatingpurine derivative.
 6. The method of claim 1 wherein said mammaliandisease condition is Alzheimer's disease and related degenerativedisorders.
 7. The method of claim 1 wherein said mammalian diseasecondition is old age benign forgetfulness and related disorders.
 8. Themethod of claim 1 wherein said mammalian disease condition is agingrelated loss of neurons or neuronal connectivity and relateddeterioration of sensory, motor, reflex, or cognitive abilities.
 9. Themethod of claim 1 wherein said mammalian disease condition isParkinson's disease and related disorders.
 10. The method of claim 1wherein said mammalian disease condition is spino-cerebellar atrophy.11. The method of claim 1 wherein said mammalian disease condition ismotor neuronopathy or Amyotrophic Lateral Sclerosis.
 12. The method ofclaim 1 wherein said mammalian disease condition is damage to neurons ortheir processes by physical agents.
 13. The method of claim 1 whereinsaid mammalian disease condition is damage to neurons by ischemia,anoxia, hypoxia, or hypoglycemia.
 14. The method of claim 1 wherein saidmammalian disease condition is damage to neurons by chemical agents. 15.The method of claim 1 wherein said mammalian disease condition is traumato the heart, brain or spinal cord.
 16. The method of claim 1 whereinsaid mammalian disease condition is epilepsy or seizures.
 17. The methodof claim 1 wherein said mammalian disease condition is peripheralneuropathy.
 18. The method of claim 1 wherein said mammalian diseasecondition is learning disability.
 19. The method of claim 1 wherein saidmammalian disease condition is cerebral palsy.
 20. The method of claim 1wherein said mammalian disease condition is psychiatric disorder. 21.The method of claim 1 wherein said mammalian disease condition is memorydisorder.
 22. The method of claim 1 wherein said mammalian diseasecondition is Huntington's disease.
 23. The method of claim 1 whereinsaid endogenous antioxidant is a bile pigment.
 24. The method of claim23 wherein said bile pigment is selected from the group consisting ofbiliverdin or bilirubin.
 25. The method of claim 23 wherein said bilepigment is produced through the additional step of degrading heme withheme oxygenase.
 26. A method for inducing the in vivo production of hemeoxygenase in a mammal, said method comprising the step of treating amammal with an effective amount of at least one carbon monoxidedependent guanylyl cyclase modulating purine derivative.
 27. The methodof claim 26 wherein said carbon monoxide dependent guanylyl cyclasemodulating purine derivative is selected from the group consisting ofguanosine,4-[[3-(1,6-dihydro-6-oxo-9H-purin-9-yl)-1-oxo-propyl]amino]benzoic acid,and inosine pranobex.
 28. The method of claim 26 wherein said effectiveamount of said at least one carbon monoxide dependent guanylyl cyclasemodulating purine derivative produces a treating concentration of atleast 1 μM.
 29. The method of claim 27 wherein said mammal is treated byorally administering said at least one carbon monoxide dependentguanylyl cyclase modulating purine derivative.
 30. The method of claim27 wherein said mammal is treated by injecting said at least one carbonmonoxide dependent guanylyl cyclase modulating purine derivative. 31.The method of claim 26 further comprising the additional step ofinducing the degrading of heme in said mammal with said heme oxygenaseto endogenously produce bile pigment and carbon monoxide.
 32. The methodof claim 31 further comprising the additional step of modulatingguanylyl cyclase in said mammal with said endogenously produced carbonmonoxide.
 33. The method of claim 31 further comprising the additionalstep of neutralizing or sequestering free radicals in said mammal withsaid endogenously produced bile pigment.
 34. The method of claim 31further comprising the additional step of inducing a reduction in theblood pressure of said mammal with said endogenously produced carbonmonoxide.